Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

S-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking S-palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (Cys1169, Cys1170, and Cys1978) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys1978 is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys1978 S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions.

The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance.

Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton–Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle–dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.

Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.

(American Association for the Advancement of Science) Microplastic particles entering the sea surface were thought to settle to the seafloor directly below them, but now, a new study reveals that slow-moving currents near the bottom of the ocean direct the flow of plastics, creating microplastic hotpots in sediments of the deep sea.

(Damon Runyon Cancer Research Foundation) The first class of Damon Runyon Quantitative Biology Fellowship Awardees launched their research in novel directions that may lead to the next breakthroughs in cancer research. Nine brilliant young scientists will apply their quantitative skills to design innovative experiments and interpret massive data sets that may help solve important biological and clinical problems.

(University of Exeter) The public would comply with major changes to medical advice - but would then be less likely to follow other new guidelines in the future, research shows.

(University of Tsukuba) Researchers from the University of Tsukuba discovered that fish schools showed a significant change in behavior with varying school sizes. Using integrated information theory, they showed that a significant change in the interaction between the fish and the overall collective behavior occurred between three- and four-fish schools, including the emergence of leadership within the group. These findings help understand the dynamics of collective behavior.

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance.

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

Payman Samavarchi-Tehrani
May 1, 2020; 19:757-773
Review

Invitation Only Research Event

Objective: To investigate the lesion detection rate of ¹⁸F-DCFPyL-PET/CT, a prostate-specific membrane antigen (PSMA) targeted PET agent, in biochemical relapse prostate cancer patients after primary local therapy. Methods: This is a prospective institutional review board-approved study of 90 patients with documented biochemical recurrence (median PSA 2.5 ng/mL, range 0.21-35.5 ng/mL) with negative conventional imaging after primary local therapies, including radical prostatectomy (n = 38), radiation (n = 27) or combination (n = 25). Patients on androgen deprivation therapy were excluded. Patients underwent whole-body ¹⁸F-DCFPyL-PET/CT (299.9±15.5 MBq) at 2 h p.i. PSMA-PET lesion detection rate was correlated with PSA, PSA kinetics and original primary tumor grade. Results: Seventy patients (77.8%) showed a positive PSMA-PET scan, identifying a total of 287 lesions: 37 prostate bed foci, 208 lymph nodes, and 42 bone/organ distant sites; 11 patients had a negative scan and 9 patients showed indeterminate lesions, which were considered negative in this study. The detection rates were 47.6% (n = 10/21), 50% (n = 5/10), 88.9% (n = 8/9), and 94% (n = 47/50) for PSA >0.2 to <0.5, 0.5 to <1.0, 1 to <2.0, and ≥2.0 ng/mL, respectively. In post-surgical patients, PSA, PSAdt and PSAvel correlated with PET results but the same was not true for post-radiation patients. These parameters also correlated with the extent of disease on PET (intrapelvic vs. extrapelvic). There was no significant difference between the rate of positive scans in patients with higher grade vs lower grade primary tumors (Gleason score ≥4+3 vs <3+4). Tumor recurrence was histology confirmed in 40% (28/70) of patients. On a per-patient basis, positive predictive value was 93.3% (95% CI, 77.6-99.2%) by histopathologic validation, and 96.2% (95% CI, 86.3-99.7%) by the combination of histology and imaging/clinical follow-up. Conclusion: ¹⁸F-DCFPyL-PET/CT imaging offers high detection rates in biochemically recurrent prostate cancer patients; and is positive in about 50% of patients with PSA <0.5 ng/mL, which could substantially impact clinical management. In post-surgical patients, ¹⁸F-DCFPyL-PET/CT correlates with PSA, PSAdt and PSAvel suggesting it may have prognostic value. ¹⁸F-DCFPyL-PET/CT is highly promising for localizing sites of recurrent prostate cancer.

Background: ⁶⁸Ga PSMA PET CT (PSMA) is increasingly used in men with biochemical recurrence (BCR) post radical prostatectomy (RP), but its longer term prognostic / predictive potential in these men is unknown. The aim of this study was to evaluate the predictive value of PSMA PET for 3 year freedom from progression (FFP) in men with BCR post RP undergoing salvage radiotherapy (sRT). Methods: This prospective multi-center study enrolled 260 men between 2015 and 2017. Eligible patients were referred for PSMA with rising PSA following RP. Management following PSMA was recorded but not mandated. PSMA protocols were standardised across sites and reported prospectively. Clinical, pathological and surgical information, sRT, timing and duration of androgen deprivation (ADT), 3 year PSA results and clinical events were documented. FFP was defined as a PSA rise ≤ 0.2ng/mL above nadir post sRT, with no additional treatment. Results: The median PSA was 0.26ng/mL (IQR 0.15 - 0.59) and follow-up 38 months (IQR 31-43). PSMA was negative in 34.6% (90/260), confined to prostate fossa 21.5% (56/260), pelvic nodes 26.2% (68/260), and distant disease 17.7% (46/260). 71.5% (186/260) received sRT, 38.2% (71/186) to the fossa only, 49.4% (92/186) fossa + pelvic nodes and 12.4% (23/186) nodes alone/SBRT. PSMA was highly predictive of FFP at 3 years following sRT. Overall, FFP was achieved in 64.5% (120/186) of those who received sRT, 81% (81/100) with negative/fossa confined vs. 45% (39/86) for extra fossa disease (p<0.0001). On logistic regression PSMA was more independently predictive of FFP than established clinical predictors, including PSA, T-stage, surgical margin status or Gleason score (P < 0.002). 32% of men with a negative PSMA PET did not receive treatment. Of these, 66% (19/29) progressed, with a mean rise in PSA of 1.59ng/mL over the 3 years. Conclusion: PSMA PET result is highly predictive of FFP at 3 years in men undergoing sRT for BCR following RP. In particular, men with negative PSMA PET or disease identified as still confined to the prostate fossa demonstrate high FFP, despite receiving less extensive radiotherapy and lower rates of additional ADT than those with extra fossa disease.

Background: Targeting cancer-associated fibroblasts (CAFs) has become an attractive goal for diagnostic imaging and therapy as they can constitute as much as 90% of tumor mass. The serine protease fibroblast activation protein (FAP) is overexpressed selectively in CAFs, drawing interest in FAP as a stromal target. The quinoline-based FAP-inhibitor PET tracer, ⁶⁸Ga-FAPI-04, has been previously shown to yield high tumor-to-background ratios (TBR) in patients with various cancers. Recent developments towards an improved compound for therapeutic application have identified FAPI-46 as a promising agent due to a longer tumor retention time in comparison with FAPI-04. Here we present a PET biodistribution and radiation dosimetry study of ⁶⁸Ga-FAPI-46 in cancer patients. Methods: Six patients with different cancers underwent serial ⁶⁸Ga-FAPI-46 PET/CT scans at three time points following radiotracer injection: 10 minutes, 1 hour, and 3 hours. The source organs consisted of the kidneys, bladder, liver, heart, spleen, bone marrow, uterus, and body remainder. OLINDA/EXM v.1.1 software was used to fit and integrate the kinetic organ activity data to yield total body and organ time-integrated activity coefficients/residence times and finally organ absorbed doses. Standardized uptake values (SUV) and TBR were generated from the contoured tumor and source organ volumes. Spherical volumes in muscle and blood pool were also obtained for TBR (Tumor SUV_max / Organ SUVmean). Results: At all timepoints, the highest organ SUV_max was observed in the liver. Tumor and organ mean SUVs decreased whereas TBRs in all organs but the uterus increased with time. The highest TBRs at 3 hours were observed with the bone marrow (31.1), muscle (22.8), heart (19.1), and spleen (19.0). Organs with the highest effective doses were the bladder wall (2.41E-03 mSv/MBq), followed by ovaries (1.15E-03 mSv/MBq) and red marrow (8.49E-04mSv/MBq). The average effective total body dose was 7.80E-03 mSv/MBq. Thus for administration of 200 MBq ⁶⁸Ga-FAPI-46 the effective total body dose is 1.56 mSv ± 0.26 mSv, in addition to approximately 3.7 mSv from one low-dose CT scan done for attenuation correction. Conclusion: ⁶⁸Ga-FAPI-46 PET/CT has a favorable dosimetry profile with an estimated whole body dose of 5.3 mSv for an administration of 200 MBq (5.4 mCi) of ⁶⁸Ga-FAPI-46 (1.56± 0.26 mSv from the PET tracer and 3.7 mSv from one low-dose CT scan). The biodistribution study showed high TBRs increasing over time, suggesting high diagnostic performance and favorable tracer kinetics for potential therapeutic applications.

The purpose of this study was to investigate the feasibility and diagnostic efficacy of ⁶⁸Ga-PSMA positron emission tomography/computed tomography (PET/CT) combined with PET-ultrasound image-guided biopsy in the diagnosis of prostate cancer. Methods: A total of 31 patients with previously negative prostate biopsy, but persistent elevated serum prostate specific antigen (PSA), were imaged with a ⁶⁸Ga-labeled prostate-specific membrane antigen (PSMA) PET/CT ligand prior to undergoing repeat prostate biopsy. Based on the proposed PROMISE criteria, PSMA PET/CT results were interpreted as negative (miPSMA-ES 0-1) or positive (miPSMA-ES 2-3). All patients underwent standard template systematic biopsy with up to four additional PSMA PET-ultrasound fusion image-guided biopsy cores. The sensitivity, specificity, positive and negative predictive values, and accuracy of PSMA PET/CT were determined. In addition, the correlation between miPSMA-ES and detection rate of prostate cancer was also analyzed. Univariate logistic regression models were established using PSMA PET/CT semi-quantitative analysis parameters to predict the outcome of repeat prostate biopsy. Results: The median age of patients was 65 years (range 53-81), and the median PSA level was 18.0 ng/ml (range 5.48-49.77 ng/ml). Prostate cancer was detected in 15/31 patients (48.4%) and 12/31 patients (38.7%) had clinically significant disease. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of ⁶⁸Ga-PSMA PET/CT in the diagnosis of clinically significant prostate cancer were 100.0%, 68.4%, 66.7%, 100.0% and 80.6%, respectively. The detection rate of prostate cancer increased with the increase of miPSMA-ES score. The detection rate of clinically significant prostate cancer in miPSMA-ES 0-1, 2 and 3 groups were 0%, 54.5% and 85.7% respectively. Semi-quantitative analysis of ⁶⁸Ga-PSMA PET/CT images showed that predictive models based on maximum standardized uptake value (SUV_max), tumor-to-background normal prostate SUV (SUVT/BGp) and tumor-to-background normal liver SUV (SUVratio) could effectively predict clinically significant prostate cancer; area under the curves were 0.930, 0.877, and 0.956, respectively. Conclusion: This study preliminarily confirmed that ⁶⁸Ga-PSMA PET/CT imaging combined with PET-ultrasound fusion image-guided prostate biopsy can effectively detect clinically significant prostate cancer. Prebiopsy ⁶⁸Ga-PSMA PET/CT has predictive value for clinically significant cancer in the studied patient population.

We reviewed ¹¹¹In-DOTA-anti-CD45 antibody (BC8) imaging and bone marrow biopsy measurements to ascertain biodistribution and biokinetics of the radiolabeled antibody and to investigate differences based on type of hematologic malignancy. Methods: Serial whole-body scintigraphic images (4 time-points) were obtained after infusion of the ¹¹¹In-DOTA-BC8 (176-406 MBq) in 52 adult patients with hematologic malignancies (lymphoma, multiple myeloma, acute myeloid leukemia and myelodysplastic syndrome). Counts were obtained for the regions of interest for spleen, liver, kidneys, testicles (in males), and two marrow sites (acetabulum and sacrum) and correction for attenuation and background was made. Bone marrow biopsies were obtained 14-24 hours post-infusion and percent of administered activity was determined. Radiation absorbed doses were calculated. Results: Initial uptake in liver averaged 32% ± 8.4% (S.D.) of administered activity (52 patients), which cleared monoexponentially with biological half-time of 293 ± 157 hours (33 patients) or did not clear (19 patients). Initial uptake in spleen averaged 22% ± 12% and cleared with a biological half-time 271 ± 185 hours (36 patients) or longer (6 patients). Initial uptake in kidney averaged 2.4% ± 2.0% and cleared with a biological half-time of 243 ± 144 hours (27 patients) or longer (9 patients). Initial uptake in red marrow averaged 23% ± 11% and cleared with half-times of 215 ± 107 hours (43 patients) or longer (5 patients). Whole-body retention half-times averaged 198 ± 75 hours. Splenic uptake was higher in the AML/MDS group when compared to the lymphoma group (p ≤ 0.05) and to the multiple myeloma group (p ≤ 0.10). Liver represented the dose-limiting organ. For liver uptake, no significant differences were observed between the three malignancy groups. Average calculated radiation absorbed doses per unit administered activity for a therapy infusions of ⁹⁰Y-DOTA-BC8 were for red marrow: 470 ± 260 cGy/MBq, liver 1100 ± 330 cGy/MBq, spleen 4120 ± 1950 cGy/MBq, total body 7520 ± 20 cGy/MBq, osteogenic cells 290 ± 200 cGy/MBq, and kidneys 240 ± 200 cGy/MBqR. Conclusion: ¹¹¹In-DOTA-BC8 had long retention time in liver, spleen, kidneys, and red marrow, and the highest absorbed doses were calculated for spleen and liver. Few differences were observed by malignancy type. The exception was greater splenic uptake among leukemia/MDS group when compared to lymphoma and multiple myeloma groups.

For individual treatment decisions in patients with metastatic prostate cancer (mPC), molecular diagnostics are increasingly used. Bone metastases are frequently the only source for obtaining metastatic tumor tissue. However, the success rate of computed tomography (CT)-guided bone biopsies for molecular analyses in mPC patients is only ~40%. Positron emission tomography (PET) using Gallium-68 prostate specific membrane antigen (⁶⁸Ga-PSMA) is a promising tool to improve the harvest rate of bone biopsies for molecular analyses. Aim of this study was to determine the success rate of ⁶⁸Ga-PSMA guided bone biopsies for molecular diagnostics in mPC patients. Methods: Within a prospective multicenter whole-genome sequencing trial (NCT01855477), 69 mPC patients underwent ⁶⁸Ga-PSMA PET/CT prior to bone biopsy. Primary endpoint was success rate (tumor percentage ≥30%) of ⁶⁸Ga-PSMA guided bone biopsies. At biopsy sites, ⁶⁸Ga-PSMA uptake was quantified using rigid body image registration of ⁶⁸Ga-PSMA PET/CT and interventional CT. Actionable somatic alterations were identified. Results: Success rate of ⁶⁸Ga-PSMA guided biopsies for molecular analyses was 70%. At biopsy sites categorized as positive, inconclusive, or negative for ⁶⁸Ga-PSMA uptake, 70%, 64%, and 36% of biopsies were tumor positive (≥30%), respectively (P = 0.0610). In tumor positive biopsies, ⁶⁸Ga-PSMA uptake was significantly higher (P = 0.008), whereas radiodensity was significantly lower (P = 0.006). With an area under the curve of 0.84 and 0.70, both ⁶⁸Ga-PSMA uptake (maximum standardized uptake value) and radiodensity (mean Hounsfield Units) were strong predictors for a positive biopsy. Actionable somatic alterations were detected in 73% of the sequenced biopsies. Conclusion: In patients with mPC, ⁶⁸Ga-PSMA PET/CT improves the success rate of CT-guided bone biopsies for molecular analyses, thereby identifying actionable somatic alterations in more patients. Therefore, ⁶⁸Ga-PSMA PET/CT may be considered for guidance of bone biopsies in both clinical practice and clinical trials.

BiTE® (Bispecific T-cell engager) molecules are designed to engage and activate cytotoxic T-cells to kill tumor cells. Little is known about their biodistribution in immunocompetent settings. To explore their pharmacokinetics and the role of the immune cells, BiTE molecules were radiolabeled with positron emission tomography (PET) isotope zirconium-89 (89Zr) and studied in immunocompetent and immunodeficient mouse models. PET images and ex-vivo biodistribution in immunocompetent mice with 89Zr-muS110, targeting mouse CD3 (Kd = 2.9 nM) and mouse EpCAM (Kd = 21 nM), and 89Zr-hyS110, targeting only mouse CD3 (Kd = 2.9 nM), showed uptake in tumor, spleen and other lymphoid organs, while the human-specific control BiTE 89Zr-AMG 110 showed similar tumor uptake but lacked spleen uptake. 89Zr-muS110 spleen uptake was lower in immunodeficient than in immunocompetent mice. After repeated administration of non-radiolabeled muS110 to immunocompetent mice 89Zr-muS110 uptake in spleen, and other lymphoid tissues, decreased and was comparable to uptake in immunodeficient mice, indicating saturation of CD3 binding sites. Autoradiography and immunohistochemistry demonstrated colocalization of 89Zr-muS110 and 89Zr-hyS110 with CD3-positive T-cells in the tumor and spleen but not with EpCAM expression. Also, uptake in the duodenum correlated with a high incidence of T-cells. This study shows that in immunocompetent mice the BiTE 89Zr-muS110 distribution is predominantly based on its high affinity CD3 binding arm. Significance: 89Zr-muS110 biodistribution is mainly dependent on the T-cell targeting arm with limited contribution of its second arm, targeting EpCAM. These findings highlight the need for extensive biodistribution studies of novel bispecific constructs as results might have implications for their respective drug development and clinical translation.

Prostate specific membrane antigen (PSMA) targeting Positron Emission Tomography (PET) imaging is becoming the reference standard for prostate cancer (PC) staging, especially in advanced disease. Yet, the implications of PSMA-PET derived whole-body tumor volume for overall survival are poorly elucidated to date. This might be due to the fact that (semi-) automated quantification of whole-body tumor volume as PSMA-PET biomarker is an unmet clinical challenge. Therefore, a novel semi-automated software is proposed and evaluated by the present study, which enables the semi-automated quantification of PSMA-PET biomarkers such as whole-body tumor volume. Methods: The proposed quantification is implemented as a research prototype (MI Whole Body Analysis Suite, v1.0, Siemens Medical Solutions USA, Inc., Knoxville, TN). PSMA accumulating foci were automatically segmented by a percental threshold (50% of local SUV_max). Neural networks were trained to segment organs in PET-CT acquisitions (training CTs: 8,632, validation CTs: 53). Thereby, PSMA foci within organs of physiologic PSMA uptake were semi-automatically excluded from the analysis. Pretherapeutic PSMA-PET-CTs of 40 consecutive patients treated with ¹⁷⁷Lu-PSMA-617 therapy were evaluated in this analysis. The volumetric whole-body tumor volume (PSMATV50), SUV_max, SUVmean and other whole-body imaging biomarkers were calculated for each patient. Semi-automatically derived results were compared with manual readings in a sub-cohort (by one nuclear medicine physician using syngo.MM Oncology software, Siemens Healthineers, Knoxville, TN). Additionally, an inter-observer evaluation of the semi-automated approach was performed in a sub-cohort (by two nuclear medicine physicians). Results: Manually and semi automatically derived PSMA metrics were highly correlated (PSMATV50: R2=1.000; p<0.001; SUV_max: R2=0.988; p<0.001). The inter-observer agreement of the semi-automated workflow was also high (PSMATV50: R2=1.000; p<0.001; ICC=1.000; SUV_max: R2=0.988; p<0.001; ICC=0.997). PSMATV50 [ml] was a significant predictor of overall survival (HR: 1.004; 95%CI: 1.001-1.006, P = 0.002) and remained so in a multivariate regression including other biomarkers (HR: 1.004; 95%CI: 1.001-1.006 P = 0.004). Conclusion: PSMATV50 is a promising PSMA-PET biomarker that is reproducible and easily quantified by the proposed semi-automated software. Moreover, PSMATV50 is a significant predictor of overall survival in patients with advanced prostate cancer that receive ¹⁷⁷Lu-PSMA-617 therapy.

The purpose of this study was to establish the dose-response relationship of selective internal radiation therapy (SIRT) in patients with metastatic colorectal cancer (mCRC), when informed by radiobiological sensitivity parameters derived from mCRC cell lines exposed to yttrium-90 (⁹⁰Y). Methods: 23 mCRC patients with liver metastases refractory to chemotherapy were included. ⁹⁰Y bremsstrahlung SPECT images were transformed into dose maps assuming the local dose deposition method. Baseline and follow-up CT scans were segmented to derive liver and tumor volumes. Mean, median, and D₇₀ (minimum dose to 70% of tumor volume) values determined from dose maps were correlated with change in tumor volume and vRECIST response using linear and logistic regression, respectively. Radiosensitivity parameters determined by clonogenic assays of mCRC cell lines HT-29 and DLD-1 after exposure to ⁹⁰Y or external beam radiotherapy (EBRT; 6MV photons) were used in biological effective dose (BED) calculations. Results: Mean administered radioactivity was 1469±428 MBq (847-2185 MBq), achieving a mean radiation absorbed tumor dose of 35.5±9.4 Gy and mean normal liver dose of 26.4±6.8 Gy. A 1.0 Gy increase in mean, median, and D₇₀ absorbed dose was associated with reduction in tumor volume of 1.8%, 1.8%, and 1.5%, respectively, and increased probability of vRECIST response (odds ratio: 1.09, 1.09, and 1.10 respectively). Threshold mean, median and D₇₀ doses for response were 48.3, 48.8, and 41.8 Gy respectively. EBRT-equivalent BEDs for ⁹⁰Y are up to 50% smaller than those calculated by applying protraction-corrected radiobiological parameters derived from EBRT alone. Conclusion: Dosimetric studies have assumed equivalence between ⁹⁰Y SIRT and EBRT, leading to inflation of BED for SIRT and possible under-treatment. Radiobiological parameters for ⁹⁰Y were applied to a BED model, providing a calculation method that has the potential to improve assessment of tumor control.

Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERβ) has been found to be expressed in a fraction of TNBCs where, because of its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERβ under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERβ in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERβ of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERβ association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodeling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERβ association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERβ in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodeling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.

The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Chronic hyperglycemia is known to disrupt the proteolytic milieu, initiating compensatory and maladaptive pathways in the diabetic kidney. Such changes in intrarenal proteolysis are captured by the urinary peptidome. To elucidate the early kidney response to chronic hyperglycemia, we conducted a peptidomic investigation into urines from otherwise healthy youths with type 1 diabetes and their non-diabetic peers using unbiased and targeted mass spectrometry-based techniques. This cross-sectional study included two separate cohorts for the discovery (n = 30) and internal validation (n = 30) of differential peptide excretion. Peptide bioactivity was predicted using PeptideRanker and subsequently verified in vitro. Proteasix and the Nephroseq database were used to identify putative proteases responsible for peptide generation and examine their expression in diabetic nephropathy. A total of 6550 urinary peptides were identified in the discovery analysis. We further examined the subset of 162 peptides, which were quantified across all thirty samples. Of the 15 differentially excreted peptides (p < 0.05), seven derived from a C-terminal region (⁵⁸⁹SGSVIDQSRVLNLGPITRK⁶⁰⁷) of uromodulin, a kidney-specific protein. Increased excretion of five uromodulin peptides was replicated in the validation cohort using parallel reaction monitoring (p < 0.05). One of the validated peptides (SGSVIDQSRVLNLGPI) activated NFB and AP-1 signaling, stimulated cytokine release, and enhanced neutrophil migration in vitro. In silico analyses highlighted several potential proteases such as hepsin, meprin A, and cathepsin B to be responsible for generating these peptides. In summary, we identified a urinary signature of uromodulin peptides associated with early type 1 diabetes before clinical manifestations of kidney disease and discovered novel bioactivity of uromodulin peptides in vitro. Our present findings lay the groundwork for future studies to validate peptide excretion in larger and broader populations, to investigate the role of bioactive uromodulin peptides in high glucose conditions, and to examine proteases that cleave uromodulin.

Lung cancer (LC) remains the leading cause of mortality from malignant tumors worldwide. In our previous study, we surveyed both IgG and IgM-bound serological biomarkers and validated a panel of IgG-bound autoantigens for early LC diagnosis with 50% sensitivity at 90% specificity. To further improve the performance of these serological biomarkers, we surveyed HuProt arrays, comprised of 20,240 human proteins, for IgA-bound autoantigens because IgAs are a major immunoglobulin isotype in the lung. Integrating with IgG-bound autoantigens, we discovered and validated a combined biomarker panel using ELISA-format tests. Specifically, in Phase I, we obtained IgA-based autoimmune profiles of 69 early stage LC patients, 30 healthy subjects and 25 patients with lung benign lesions (LBL) on HuProt arrays and identified 28 proteins as candidate autoantigens that were significantly associated with early stage LC. In Phase II, we re-purified the autoantigens and converted them into an ELISA-format testing to profile an additional large cohort, comprised of 136 early stage LC patients, 58 healthy individuals, and 29 LBL patients. Integration of IgG autoimmune profiles allowed us to identify and validate a biomarker panel of three IgA autoantigens (i.e. BCL7A, and TRIM33 and MTERF4) and three IgG autoantigens (i.e. CTAG1A, DDX4 and MAGEC2) for diagnosis of early stage LC with 73.5% sensitivity at >85% specificity. In Phase III, the performance of this biomarker panel was confirmed with an independent cohort, comprised of 88 early stage LC patients, 18 LBL patients, and 36 healthy subjects. Finally, a blind test on 178 serum samples was conducted to confirm the performance of the biomarker panel. In summary, this study demonstrates for the first time that an integrated panel of IgA/IgG autoantigens can serve as valuable biomarkers to further improve the performance of early diagnosis of LC.

The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.