Ileana M. Cristea
Dec 1, 2005; 4:1933-1941
Research

M. Wang
Aug 1, 2012; 11:492-500
Technological Innovation and Resources

Jonathan C. Trinidad
Aug 1, 2012; 11:215-229
Research

Mitsunori Fukuda
Jun 1, 2008; 7:1031-1042
Research

Hasmik Keshishian
Dec 1, 2007; 6:2212-2229
Research

Qin Liu
Aug 1, 2007; 6:1299-1317
Research

Tamar Geiger
Mar 1, 2012; 11:M111.014050-M111.014050
Special Issue: Prospects in Space and Time

Yi Zhang
Sep 1, 2005; 4:1240-1250
Research

Timothy J. Griffin
Apr 1, 2002; 1:323-333
Research

Lawrence E. Ostrowski
Jun 1, 2002; 1:451-465
Research

Sean R. Collins
Mar 1, 2007; 6:439-450
Research

Sebastian A. Wagner
Oct 1, 2011; 10:M111.013284-M111.013284
Research

Christian Tagwerker
Apr 1, 2006; 5:737-748
Research

Joris J. Benschop
Jul 1, 2007; 6:1198-1214
Research

Guoan Chen
Apr 1, 2002; 1:304-313
Research

Qiang Tian
Oct 1, 2004; 3:960-969
Research

Jesper V. Olsen
Jun 1, 2004; 3:608-614
Technology

Alexey I. Nesvizhskii
Oct 1, 2005; 4:1419-1440
Tutorial

Ludovic C. Gillet
Jun 1, 2012; 11:O111.016717-O111.016717
Research

Yu Xue
Sep 1, 2008; 7:1598-1608
Research

Claudio P. Albuquerque
Jul 1, 2008; 7:1389-1396
Research

Mathias Uhlén
Dec 1, 2005; 4:1920-1932
Research

William M. Old
Oct 1, 2005; 4:1487-1502
Research

Leigh Anderson
Apr 1, 2006; 5:573-588
Research

Ignat V. Shilov
Sep 1, 2007; 6:1638-1655
Technology

Albrecht Gruhler
Mar 1, 2005; 4:310-327
Research

Martin R. Larsen
Jul 1, 2005; 4:873-886
Technology

Jeffrey C. Silva
Jan 1, 2006; 5:144-156
Research

N. Leigh Anderson
Nov 1, 2002; 1:845-867
Reviews/Perspectives

Linn Fagerberg
Feb 1, 2014; 13:397-406
Research

Ulrich Rothbauer
Feb 1, 2008; 7:282-289
Research

Yasushi Ishihama
Sep 1, 2005; 4:1265-1272
Research

Jesper V. Olsen
Dec 1, 2005; 4:2010-2021
Technology

Eiji Kinoshita
Apr 1, 2006; 5:749-757
Technology

Philip L. Ross
Dec 1, 2004; 3:1154-1169
Research

Jürgen Cox
Sep 1, 2014; 13:2513-2526
Technological Innovation and Resources

Shao-En Ong
May 1, 2002; 1:376-386
Research

In our computational study, we use molecular simulations to substantiate a hypothetical mechanism for glycosidic bond cleavage in the presence of a single catalytic acid at the active site of the mutant D10N HiCel45A. In addition to discussing this plausible mechanism from the context of structurally related MltA lytic transglycosylase and subfamily C GH45s, we also suggest the implications of the plausible mechanism for our current understanding of the action of expansins and lytic transglycosylases. As correctly pointed out by Professor Cosgrove (1), there is large body of evidence, a significant portion of which was regrettably not discussed in our paper, that suggests that expansins are incapable of lytic action on polysaccharide substrates. Whereas these insights do not change the results or the conclusions of our article, we would like to thank Professor Cosgrove for these additional insights. In particular, our main point with respect to expansins is that our results suggest the possibility that expansins are capable of nonhydrolytic lytic activity. Our intention was not to suggest this was the mechanism of expansins, but that it should be considered based on our results and the similarity of the active sites.The molecular mechanisms of how expansins enable cell wall expansion remains to be fully understood. Whereas our proposed mechanism resulting in the formation of the 1,6-anhdro product might be found in expansins and might contribute to the mode of action of expansins, we would like to emphasize that the intent of this study was only to suggest this as a...

From their simulations of endoglucanase Cel45A, Bharadwaj et al. (1) propose that structurally related expansins and MltA may cut glycan backbones without generating reducing ends. This is tenable for MltA, a peptidoglycan lytic transglycosylase whose action produces nonreducing 1,6-anhydro products, but is untenable for expansins.Expansins loosen plant cell walls and induce wall expansion. Contrary to the assertion by Bharadwaj et al., the conclusion that expansins are not lytic is not merely based on lack of new reducing ends but is supported by multiple (negative) tests for polysaccharide cleavage that do not rely on detection of reducing ends. At least eight studies with three divergent groups of expansins document this point. For instance, α-expansin did not reduce the viscosity of various wall polysaccharide solutions, an endolytic assay that does not rely on measuring reducing ends (e.g. see Ref. 2 and other studies).Walls treated with α-expansin did not release saccharide fragments, measured by pulsed amperometric detection, which can detect nonreducing saccharides (3).In the case of β-expansins, protein treatments did not cleave the backbones of a wide range of dye-coupled cross-linked wall polysaccharides; nor did they cleave backbones of polysaccharides extracted from plant cell walls, measured by gel permeation chromatography (4).For five microbial expansins, tests with a range of dye-coupled cross-linked polysaccharides likewise did not detect lytic activity (e.g. see Ref. 5). Thus, extensive published evidence argues against lytic action by expansins, as proposed by Bharadwaj (1), and attempts to identify 1,6-anhydro products seem unlikely to succeed.

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.

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.

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

Hypersecretion of glucagon from pancreatic α-cells strongly contributes to diabetic hyperglycemia. Moreover, failure of α-cells to increase glucagon secretion in response to falling blood glucose concentrations compromises the defense against hypoglycemia, a common complication in diabetes therapy. However, the mechanisms underlying glucose regulation of glucagon secretion are poorly understood and likely involve both α-cell–intrinsic and intraislet paracrine signaling. Among paracrine factors, glucose-stimulated release of the GABA metabolite γ-hydroxybutyric acid (GHB) from pancreatic β-cells might mediate glucose suppression of glucagon release via GHB receptors on α-cells. However, the direct effects of GHB on α-cell signaling and glucagon release have not been investigated. Here, we found that GHB (4–10 μm) lacked effects on the cytoplasmic concentrations of the secretion-regulating messengers Ca2+ and cAMP in mouse α-cells. Glucagon secretion from perifused mouse islets was also unaffected by GHB at both 1 and 7 mm glucose. The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1 mm. Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mm glucose and did not prevent the suppressive effect of 7 mm glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mm glucose, an effect mimicked by 3-chloropropanoic acid. We conclude that GHB does not mediate the inhibitory effect of glucose on glucagon secretion.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

The linear ubiquitin assembly complex (LUBAC) is an essential component of the innate and adaptive immune system. Modification of cellular substrates with linear polyubiquitin chains is a key regulatory step in signal transduction that impacts cell death and inflammatory signaling downstream of various innate immunity receptors. Loss-of-function mutations in the LUBAC components HOIP and HOIL-1 yield a systemic autoinflammatory disease in humans, whereas their genetic ablation is embryonically lethal in mice. Deficiency of the LUBAC adaptor protein Sharpin results in a multi-organ inflammatory disease in mice characterized by chronic proliferative dermatitis (cpdm), which is propagated by TNFR1-induced and RIPK1-mediated keratinocyte cell death. We have previously shown that caspase-1 and -11 promoted the dermatitis pathology of cpdm mice and mediated cell death in the skin. Here, we describe a reciprocal regulation of caspase-1 and LUBAC activities in keratinocytes. We show that LUBAC interacted with caspase-1 via HOIP and modified its CARD domain with linear polyubiquitin and that depletion of HOIP or Sharpin resulted in heightened caspase-1 activation and cell death in response to inflammasome activation, unlike what is observed in macrophages. Reciprocally, caspase-1, as well as caspase-8, regulated LUBAC activity by proteolytically processing HOIP at Asp-348 and Asp-387 during the execution of cell death. HOIP processing impeded substrate ubiquitination in the NF-κB pathway and resulted in enhanced apoptosis. These results highlight a regulatory mechanism underlying efficient apoptosis in keratinocytes and provide further evidence of a cross-talk between inflammatory and cell death pathways.

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

ATP plays important roles outside the cell, but the mechanism by which it is arrives in the extracellular environment is not clear. Dunn et al. now show that decreases in cellular cholesterol levels mediated by the ABCG1 transporter increase ATP release by volume-regulated anion channels under hypotonic conditions. Importantly, these results may imply that cells that handle cholesterol differently might experience differential extracellular ATP release during hypotonicity.

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and short hairpin RNA–based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel–dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.

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.

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge–swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge–swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal–regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.