Chi-Liang Eric Yen
Nov 1, 2008; 49:2283-2301
Thematic Reviews

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein–coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq1 isoform protein playing a major role in fly phototransduction. However, Gαq1 null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. Here, we isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, we found that mutations in the Gαq gene disrupt light responses and demonstrate that the Gαq3 isoform protein is responsible for the residual light response in Gαq1 null mutants. Moreover, we report that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. Our findings clarify essential roles of several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis.

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from ¹³C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K₂ subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Hazy CEO Harry Keen explains why artificially produced data is gaining favour over information generated by real-world events

Howard Craig Zisser
Apr 1, 2007; 20:85-89
Articles

Guillermo E. Umpierrez
Jan 1, 2002; 15:
Articles

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.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

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.

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein–coupled receptors to intracellular effector molecules. In Drosophila, the Gαq gene encodes several Gαq splice variants, with the Gαq1 isoform protein playing a major role in fly phototransduction. However, Gαq1 null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. Here, we isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, we found that mutations in the Gαq gene disrupt light responses and demonstrate that the Gαq3 isoform protein is responsible for the residual light response in Gαq1 null mutants. Moreover, we report that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. Our findings clarify essential roles of several different Gαq splice variants in phototransduction and retinal integrity in Drosophila and reveal that Gαq3 functions in rhodopsin synthesis.

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.

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.

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

(Arizona State University) Hydrogen is an essential commodity with over 60 million tons produced globally every year. However over 95 percent of it is made by steam reformation of fossil fuels, a process that is energy intensive and produces carbon dioxide. If we could replace even a part of that with algal biohydrogen that is made via light and water, it would have a substantial impact.

Guillermo E. Umpierrez
Jan 1, 2002; 15:
Articles

21 January 2020

Discussions of North African integration have evoked ideas of a shared identity and a common destiny in the region. However, recent attempts to build regional blocs in North Africa have been unsuccessful. This paper examines the benefits of a ‘synergistic’ approach to North African cooperation. 

Cri-Du-Chat Syndrome (CdCs) is a rare genetic disease caused by a deletion in the short arm of chromosome 5 (5p) with a variable clinical spectrum. To date no study in literature has ever investigated the alterations of brain glucose metabolism in these subjects by means of [¹⁸F]fluoro-2-deoxy-d-glucose Positron Emission Tomography/Computed Tomography (¹⁸F-FDG PET/CT). The aims of this study were to detect difference in brain FDG metabolism in patients affected by CdCs with different clinical presentations and identify possible "brain metabolic phenotypes" of this syndrome. Methods: 6 patients (age: 5 M and 1 F, age range: 10-27) with CdCs were assessed for presence of cognitive and behavioral symptoms with a battery of neuropsychological tests and then classified as patient with a severe or mild phenotype. Then, patients underwent a brain ¹⁸F-FDG PET/CT scan. PET/CT findings were compared to a control group, matched for age and sex, by using statistical parametric mapping (SPM). Association of different clinical phenotypes and ¹⁸F-FDG PET/CT findings was investigated. Results: Four patients presented a severe phenotype, whereas 2 patients demonstrated mild phenotype. SPM single subject and group analysis compared to the control cohort revealed a significant hypometabolism in the left temporal lobe (BAs 20, 36 and 38), in the right frontal subcallosal gyrus (BA 34) and caudate body, and in the cerebellar tonsils (p<0.001). Hypermetabolism (P = 0.001) was revealed in the right superior and precentral frontal gyrus (BA 6) in patient group compared to the control cohort. In SPM single subject analysis the hypermetabolic areas were detected only in patients with a severe phenotype. Conclusion: This study revealed different patterns of brain glucose metabolism in patients with severe and mild phenotype compared to control subjects. In particular, the hypermetabolic abnormalities in the brain, evaluated by¹⁸F-FDG PET/CT, seem to correlate with the severe phenotype in patients with CdCs.

Triple negative breast cancer (TNBC) remains the most aggressive subtype of breast cancer leading to the worst prognosis. Because current therapeutic approaches lack efficacy, there is a clinically unmet need for effective treatment alternatives. Herein, we demonstrate a promising strategy utilizing a tumor-targeting alkylphosphocholine (NM600) radiolabeled with ¹⁷⁷Lu for targeted radionuclide therapy (TRT) of TNBC. In two murine syngeneic models of TNBC, we confirmed excellent tumor targeting and rapid normal tissue clearance of the PET imaging analog ⁸⁶Y-NM600. Based on longitudinal PET/CT data acquired with ⁸⁶Y-NM600, we estimated the dosimetry of therapeutic ¹⁷⁷Lu-NM600, which showed larger absorbed doses in the tumor compared to normal tissues. Administration of ¹⁷⁷Lu-NM600 resulted in significant tumor growth inhibition and prolonged overall survival in mice bearing syngeneic 4T07 and 4T1 tumors. Complete response was attained in 60% of 4T07 bearing mice, but animals carrying aggressive 4T1 tumor grafts succumbed to metastatic progression. The injected activities used for treatment (9.25 and 18.5 MBq) were well tolerated, and only mild transient cytopenia was noted. Overall, our results suggest that ¹⁷⁷Lu-NM600 TRT has potential for treatment of TNBC and merits further exploration in a clinical setting.

Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for positron emission tomography (PET) or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in a number of malignancies including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce ⁸⁹Zr-radiolabeled onartuzumab (a monovalent, anti-human c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous ⁸⁹Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing ⁸⁹Zr-oxalate, the photoactive chelate DFO-aryl azide (DFO-ArN3) and MetMAb to give ⁸⁹ZrDFO-azepin-onartuzumab. As a control, ⁸⁹ZrDFO-Bn-NCS-onartuzumab was prepared via a conventional two-step process using pre-purified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0–72 h) was performed in female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image. Tumor specificity of ⁸⁹ZrDFO-azepin-onartuzumab was assessed by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced ⁸⁹ZrDFO-azepin-onartuzumab in <15 min. with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity (RCP) ~90% and a molar activity (Am) of ~1.5 MBq nmol^-1. Reaction optimization improved the radiochemical conversion (RCC) of ⁸⁹ZrDFO-azepin-onartuzumab to 56.9±4.1% (n = 3), with isolated RCYs of 41.2±10.6% (n = 3), and RCPs >90%. Conventional methods produced ⁸⁹ZrDFO-Bn-NCS-onartuzumab with isolated RCY >97%, RCP >97% and Am ~14.0 MBq nmol^-1. Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake reached 15.37±5.21 %ID g^-1, 6.56±4.03 %ID g^-1, respectively for ⁸⁹ZrDFO-azepin-onartuzumab (n = 4), and 21.38±11.57 %ID g^-1 and 18.84±6.03 %ID g^-1 for ⁸⁹ZrDFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34±0.47 %ID g^-1) of ⁸⁹ZrDFO-azepin-onartuzumab (n = 4). Conclusion: Experiments demonstrate that photoradiosynthesis is a viable alternative approach for producing ⁸⁹Zr-radiolabeled antibodies direct in protein formulation buffer which reduces protein aggregation and liver uptake.

McCune-Albright syndrome (MAS) is a mosaic disorder arising from gain-of-function mutations in the GNAS gene, which encodes the 3', 5'-cyclic adenosine monophosphate (cAMP) pathway-associated G-protein, Gsα. Clinical manifestations of MAS in a given individual, including fibrous dysplasia, are determined by the timing and location of the GNAS mutation during embryogenesis, the tissues involved, and the role of Gsα in the affected tissues. The Gsα mutation results in dysregulation of the cAMP signaling cascade, leading to upregulation of phosphodiesterase type 4 (PDE4), which catalyzes the hydrolysis of cAMP. Increased cAMP levels have been found in vitro in both animal models of fibrous dysplasia and in cultured cells from individuals with MAS, but not in humans with fibrous dysplasia. Positron emission tomography (PET) imaging of PDE4 with ¹¹C-(R)-rolipram has been used successfully to study the in vivo activity of the cAMP cascade. To date, it remains unknown whether fibrous dysplasia and other symptoms of MAS, including neuropsychiatric impairments, are associated with increased PDE4 activity in humans. Methods: ¹¹C-(R)-rolipram whole-body and brain PET scans were performed in six individuals with MAS (three for brain scans and six for whole-body scans) and nine healthy controls (seven for brain scans and six for whole-body scans). Results: ¹¹C-(R)-rolipram binding correlated with known locations of fibrous dysplasia in the periphery of individuals with MAS; no uptake was observed in the bones of healthy controls. In peripheral organs and the brain, no difference in ¹¹C-(R)-rolipram uptake was noted between participants with MAS and healthy controls. Conclusion: This study is the first to find evidence for increased cAMP activity in areas of fibrous dysplasia in vivo. No differences in brain uptake between MAS participants and controls were detected, which could be due to several reasons, including the limited anatomic resolution of PET. Nevertheless, the results confirm the usefulness of PET scans with ¹¹C-(R)-rolipram to indirectly measure increased cAMP pathway activation in human disease.

Fibroblast activation protein (FAP) has emerged as an interesting molecular target used in the imaging and therapy of various types of cancers. Gallium-68–labeled chelator-linked FAP inhibitors (FAPIs) have been successfully applied to positron emission tomography (PET) imaging of various tumor types. To broaden the spectrum of applicable PET tracers for extended imaging studies of FAP-dependent diseases, we herein report the radiosynthesis and preclinical evaluation of an ¹⁸F–labeled glycosylated FAP inhibitor ([¹⁸F]FGlc-FAPI). Methods: An alkyne-bearing precursor was synthesized and subjected to click chemistry–based radiosynthesis of [¹⁸F]FGlc-FAPI by two-step ¹⁸F-fluoroglycosylation. FAP-expressing HT1080hFAP cells were used to study competitive binding to FAP, cellular uptake, internalization, and efflux of [¹⁸F]FGlc-FAPI in vitro. Biodistribution studies and in vivo small animal PET studies of [¹⁸F]FGlc-FAPI compared to [⁶⁸Ga]Ga-FAPI-04 were conducted in nude mice bearing HT1080hFAP tumors or U87MG xenografts. Results: [¹⁸F]FGlc-FAPI was synthesized with a 15% radioactivity yield and a high radiochemical purity of >99%. In HT1080hFAP cells, [¹⁸F]FGlc-FAPI showed specific uptake, a high internalized fraction, and low cellular efflux. Compared to FAPI-04 (IC50 = 32 nM), the glycoconjugate, FGlc-FAPI (IC50 = 167 nM), showed slightly lower affinity for FAP in vitro, while plasma protein binding was higher for [¹⁸F]FGlc-FAPI. Biodistribution studies revealed significant hepatobiliary excretion of [¹⁸F]FGlc-FAPI; however, small animal PET studies in HT1080hFAP xenografts showed higher specific tumor uptake of [¹⁸F]FGlc-FAPI (4.5 % injected dose per gram of tissue [ID/g]) compared to [⁶⁸Ga]Ga-FAPI-04 (2 %ID/g). In U87MG tumor–bearing mice, both tracers showed similar tumor uptake, but [¹⁸F]FGlc-FAPI showed a higher tumor retention. Interestingly, [¹⁸F]FGlc-FAPI demonstrated high specific uptake in bone structures and joints. Conclusion: [¹⁸F]FGlc-FAPI is an interesting candidate for translation to the clinic, taking advantage of the longer half-life and physical imaging properties of F-18. The availability of [¹⁸F]FGlc-FAPI may allow extended PET studies of FAP-related diseases, such as cancer, but also arthritis, heart diseases, or pulmonary fibrosis.

Cerebral β-amyloid deposits and regional glucose metabolism assessed by positron emission tomography (PET) are used to distinguish between Alzheimer's disease (AD) and other dementia syndromes. In the present multicenter study, we estimated the prevalence of β-amyloid deposits on PET imaging in a wide variety of dementia syndromes and mild cognitive impairment (MCI) within a memory clinic population. Methods: Of the 1193 consecutive patients with cognitive impairment (CI) who received combined ¹⁸F-AV45 and/or ¹¹C-PIB PET, 960 were diagnosed with AD, 36 with frontotemporal dementia (FTD), 5 with dementia with Lewy bodies (DLB), 144 with MCI, 29with vascular dementia (VaD), 4 with corticobasal syndrome (CBS) and 15 with unclassifiable dementia. Baseline clinical diagnoses were independently established without access to PET imaging results. ApoE genotype analysis was performed in CI patients and 231 gender- and age-matched controls. Results: Of the 1193 CI patients, 860 (72.1%) were amyloid-positive. The prevalence of amyloid positivity in AD and MCI patients was 86.8% (833/960) and 9.7% (14/144), respectively. In FTD patients, the prevalence of β-amyloid deposits was 5.6% (2/36). In the 4 CBS patients, two were amyloid-positive. Three of the 5 DLB patients showed amyloid positivity, as did 6 of the 29 VaD (20.7%) patients. The ApoE4 allele frequency was significantly increased in amyloid-positive CI patients (30.5%) as compared with other amyloid-negative CI patients (14%) or controls (7.3%). Conclusion: Amyloid imaging may potentially be the most helpful parameter for differential diagnosis in dementia, particularly to distinguish between AD and FTD. Amyloid PET can be used in conjunction with the ApoE4 allele genetic risk test for amyloid deposits.

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.

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.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Dong-Jae Jun
May 1, 2020; 61:746-757
Research Articles

This manuscript has been withdrawn by the Author.

Plasma lipoprotein (a) [Lp(a)] levels are largely determined by variation in the LPA gene, which codes for apo(a). Genome-wide association studies (GWASs) have identified nonsynonymous variants in LPA that associate with low Lp(a) levels, although their effect on apo(a) function is unknown. We investigated two such variants, R990Q and R1771C, which were present in four null Lp(a) individuals, for structural and functional effects. Sequence alignments showed the R990 and R1771 residues to be highly conserved and homologous to each other and to residues associated with plasminogen deficiency. Structural modeling showed both residues to make several polar contacts with neighboring residues that would be ablated on substitution. Recombinant expression of the WT and R1771C apo(a) in liver and kidney cells showed an abundance of an immature form for both apo(a) proteins. A mature form of apo(a) was only seen with the WT protein. Imaging of the recombinant apo(a) proteins in conjunction with markers of the secretory pathway indicated a poor transit of R1771C into the Golgi. Furthermore, the R1771C mutant displayed a glycosylation pattern consistent with ER, but not Golgi, glycosylation. We conclude that R1771 and the equivalent R990 residue facilitate correct folding of the apo(a) kringle structure and mutations at these positions prevent the proper folding required for full maturation and secretion. To our knowledge, this is the first example of nonsynonymous variants in LPA being causative of a null Lp(a) phenotype.

Cholesteryl ester transfer protein (CETP) exists as full-length (FL) and exon 9 (E9)-deleted isoforms. The function of E9-deleted CETP is poorly understood. Here, we investigated the role of E9-deleted CETP in regulating the secretion of FL-CETP by cells and explored its possible role in intracellular lipid metabolism. CETP overexpression in cells that naturally express CETP confirmed that E9-deleted CETP is not secreted, and showed that cellular FL- and E9-deleted CETP form an isolatable complex. Coexpression of CETP isoforms lowered cellular levels of both proteins and impaired FL-CETP secretion. These effects were due to reduced synthesis of both isoforms; however, the predominate consequence of FL- and E9-deleted CETP coexpression is impaired FL-CETP synthesis. We reported previously that reducing both CETP isoforms or overexpressing FL-CETP impairs cellular triglyceride (TG) storage. To investigate this further, E9-deleted CETP was expressed in SW872 cells that naturally synthesize CETP and in mouse 3T3-L1 cells that do not. E9-deleted CETP overexpression stimulated SW872 triglyceride synthesis and increased stored TG 2-fold. Expression of E9-deleted CETP in mouse 3T3-L1 cells produced a similar lipid phenotype. In vitro, FL-CETP promotes the transfer of TG from ER-enriched membranes to lipid droplets. E9-deleted CETP also promoted this transfer, although less effectively, and it inhibited the transfer driven by FL-CETP. We conclude that FL- and E9-deleted CETP isoforms interact to mutually decrease their intracellular levels and impair FL-CETP secretion by reducing CETP biosynthesis. E9-deleted CETP, like FL-CETP, alters cellular TG metabolism and storage but in a contrary manner.

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from ¹³C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K₂ subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.