The 50 most-cited articles in Journal of Biological Chemistry, 2019

exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications-at the amino acid, peptide, or protein level-and their nature, quantity, and position…

Pathological aggregation of the transactive response DNA-binding protein of 43 kDa (TDP-43) is associated with several neurodegenerative disorders, including ALS, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer's disease. TDP-43 aggregation appears to be largely driven by its low-complexity domain (LCD), which also has a high propensity to undergo liquid-liquid phase separation (LLPS). However, the mechanism of TDP-43 LCD pathological aggregation and, most importantly, the relationship between the aggregation process and LLPS remains largely unknown. Here, we show that amyloid formation by the LCD is controlled by electrostatic repulsion. We also demonstrate that the liquid droplet environment strongly accelerates LCD fibrillation and that its aggregation under LLPS conditions involves several distinct events, culminating in rapid assembly of fibrillar aggregates that emanate from within mature liquid droplets. These combined results strongly suggest that LLPS may play a major role in pathological TDP-43 aggregation, contributing to pathogenesis in…

Gut microbial β-glucuronidase (GUS) enzymes have been suggested to be involved in the estrobolome, the collection of microbial reactions involving estrogens. Furthermore, bacterial GUS enzymes within the gastrointestinal tract have been postulated to be a contributing factor in hormone-driven cancers. However, to date, there has been no experimental evidence to support these hypotheses. Here we provide the first in vitro analysis of the ability of 35 human gut microbial GUS enzymes to reactivate two distinct estrogen glucuronides, estrone-3-glucuronide and estradiol-17-glucuronide, to estrone and estradiol, respectively. We show that certain members within the Loop 1, mini-Loop 1, and FMN-binding classes of gut microbial GUS enzymes can reactivate estrogens from their inactive glucuronides. We provide molecular details of key interactions that facilitate these catalytic processes and present the structures of two novel human gut microbial GUS enzymes related to the estrobolome. Further, we demonstrate that estrogen reactivation by Loop 1 bacterial GUS enzymes can be inhibited both…

Cryogenic electron microscopy (cryo-EM) enables structure determination of macromolecular objects and their assemblies. Although the techniques have been developing for nearly four decades, they have gained widespread attention in recent years due to technical advances on numerous fronts, enabling traditional microscopists to break into the world of molecular structural biology. Many samples can now be routinely analyzed at near-atomic resolution using standard imaging and image analysis techniques. However, numerous challenges to conventional workflows remain, and continued technical advances open entirely novel opportunities for discovery and exploration. Here, I will review some of the main methods surrounding cryo-EM with an emphasis specifically on single-particle analysis, and I will highlight challenges, open questions, and opportunities for methodology development.

To perceive their three-dimensional environment, cells and tissues must be able to sense and interpret various physical forces like shear, tensile, and compression stress. These forces can be generated both internally and externally in response to physical properties, like substrate stiffness, cell contractility, and forces generated by adjacent cells. Mechanical cues have important roles in cell fate decisions regarding proliferation, survival, and differentiation as well as the processes of tissue regeneration and wound repair. Aberrant remodeling of the extracellular space and/or defects in properly responding to mechanical cues likely contributes to various disease states, such as fibrosis, muscle diseases, and cancer. Mechanotransduction involves the sensing and translation of mechanical forces into biochemical signals, like activation of specific genes and signaling cascades that enable cells to adapt to their physical environment. The signaling pathways involved in mechanical signaling are highly complex, but numerous studies have highlighted a central role for the Hippo…

A pivotal metabolic function of insulin is the stimulation of glucose uptake into muscle and adipose tissues. The discovery of the insulin-responsive glucose transporter type 4 (GLUT4) protein in 1988 inspired its molecular cloning in the following year. It also spurred numerous cellular mechanistic studies laying the foundations for how insulin regulates glucose uptake by muscle and fat cells. Here, we reflect on the importance of the GLUT4 discovery and chronicle additional key findings made in the past 30 years. That exocytosis of a multispanning membrane protein regulates cellular glucose transport illuminated a novel adaptation of the secretory pathway, which is to transiently modulate the protein composition of the cellular plasma membrane. GLUT4 controls glucose transport into fat and muscle tissues in response to insulin and also into muscle during exercise. Thus, investigation of regulated GLUT4 trafficking provides a major means by which to map the essential signaling components that transmit the effects of insulin and exercise. Manipulation of the expression of GLUT4 or…

In the early 1980s, while using purified glycosyltransferases to probe glycan structures on surfaces of living cells in the murine immune system, we discovered a novel form of serine/threonine protein glycosylation (O-linked ␤-GlcNAc; O-GlcNAc) that occurs on thousands of proteins within the nucleus, cytoplasm, and mitochondria.Prior to this discovery, it was dogma that protein glycosylation was restricted to the luminal compartments of the secretory pathway and on extracellular domains of membrane and secretory proteins.Work in the last 3 decades from several laboratories has shown that O-GlcNAc cycling serves as a nutrient sensor to regulate signaling, transcription, mitochondrial activity, and cytoskeletal functions.O-GlcNAc also has extensive cross-talk with phosphorylation, not only at the same or proximal sites on polypeptides, but also by regulating each other's enzymes that catalyze cycling of the modifications.O-GlcNAc is generally not elongated or modified.It cycles on and off polypeptides in a time scale similar to phosphorylation, and both the enzyme that adds O-GlcNAc,…

Abstract Ferroptosis is an iron-dependent programmed cell death resulting from alterations of metabolic processes. However, its regulation and physiological significance remain to be elucidated. By analyzing transcriptional responses of murine embryonic fibroblasts exposed to the ferroptosis-inducer erastin, we found that a set of genes related to oxidative stress protection was induced upon ferroptosis. We further showed that the transcription factor BACH1 promoted ferroptosis by repressing the expression of a subset of erastin-inducible genes involved in the synthesis of glutathione or metabolism of intracellular labile iron, including Gclm, Gclc , Slc7a11 , Hmox1 , Fth1, Ftl1 , and Slc40a1 . Compared with wild-type mice, Bach1 -/- mice showed resistance to myocardial infarction, the seriousness of which was palliated by the iron-chelator deferasirox, which suppressed ferroptosis. Our findings suggest that ferroptosis is programmed at the transcriptional level to induce genes combating labile-iron-induced oxidative stress and executed upon disruption of the balance between the…

general stress response are discussed. They are also used as a basis for comparison with the general stress response in other bacteria with the aim of extracting key principles that are common among different species and highlighting important unanswered questions.

Recent studies have indicated that tau, a protein involved in Alzheimer's disease and other neurodegenerative disorders, has a propensity to undergo liquid-liquid phase separation (LLPS). However, the mechanism of this process remains unknown. Here, we demonstrate that tau LLPS is largely driven by intermolecular electrostatic interactions between the negatively charged N-terminal and positively charged middle/C-terminal regions, whereas hydrophobic interactions play a surprisingly small role. Furthermore, our results reveal that, in contrast to previous suggestions, phosphorylation is not required for tau LLPS. These findings provide a foundation for understanding the mechanism by which phosphorylation and other posttranslational modifications could modulate tau LLPS in the context of specific physiological functions as well as pathological interactions.

Phase separation of biomolecules leading to the formation of assemblies with distinct material properties has recently emerged as a new paradigm underlying subcellular organization. The discovery that disordered proteins, long associated with aggregation in neurodegenerative disease, are also implicated in driving liquid phase separation has galvanized significant interest in exploring the relationship between misregulated phase transitions and disease. This review summarizes recent work linking liquid phase separation to neurodegeneration, highlighting a pathological role for altered phase behavior and material properties of proteins assembled via liquid phase separation. The techniques that recent and current work in this area have deployed are also discussed, as is the potential for these discoveries to promote new research directions for investigating the molecular etiologies of neurodegenerative diseases.

In cortical regions of brains from individuals with preclinical or clinical Alzheimer's disease (AD), extracellular β-amyloid (Aβ) deposition precedes the aggregation of pathological intracellular tau (the product of the gene microtubule-associated protein tau (MAPT)). To our knowledge, current mouse models of tauopathy reconstitute tau pathology by overexpressing mutant human tau protein. Here, through a homologous recombination approach that replaced the entire murine Mapt gene with the human ortholog, we developed knock-in mice with humanized Mapt to create an in vivo platform for studying human tauopathy. Of note, the humanized Mapt expressed all six tau isoforms present in humans. We next cross-bred the MAPT knock-in mice with single amyloid precursor protein (App) knock-in mice to investigate the Aβ–tau axis in AD etiology. The double-knock-in mice exhibited higher tau phosphorylation than did single MAPT knock-in mice but initially lacked apparent tauopathy and neurodegeneration, as observed in the single App knock-in mice. We further observed that tau humanization…

Liquid-liquid phase separation (LLPS) facilitates the formation of condensed biological assemblies with well-delineated physical boundaries, but without lipid membrane barriers. LLPS is increasingly recognized as a common mechanism for cells to organize and maintain different cellular compartments in addition to classical membrane-delimited organelles. Membraneless condensates have many distinct features that are not present in membrane-delimited organelles and that are likely indispensable for the viability and function of living cells. Malformation of membraneless condensates is increasingly linked to human diseases. In this review, we summarize commonly used methods to investigate various forms of LLPS occurring both in 3D aqueous solution and on 2D membrane bilayers, such as LLPS condensates arising from intrinsically disordered proteins or structured modular protein domains. We then discuss, in the context of comparisons with membrane-delimited organelles, the potential functional implications of membraneless condensate formation in cells. We close by highlighting some…

Low-density lipoprotein particles are taken up by cells and delivered to the lysosome where their cholesterol esters are cleaved off by acid lipase. The released, free cholesterol is then exported from lysosomes for cellular needs or storage. This article summarizes recent advances in our understanding of the molecular basis of cholesterol export from lysosomes. Cholesterol export requires NPC intracellular cholesterol transporter 1 (NPC1) and NPC2, genetic mutations of which can cause Niemann-Pick type C disease, a disorder characterized by massive lysosomal accumulation of cholesterol and glycosphingolipids. Analysis of the NPC1 and NPC2 structures and biochemical properties, together with new structures of the related Patched (PTCH) protein, provides new clues to the mechanisms by which NPC proteins may function.

The Journal of Biological Chemistry (JBC) has been a major vehicle for disseminating and recording the discovery and characterization of proteolytic enzymes. The pace of discovery in the protease field accelerated during the 1971–2010 period that Dr. Herb Tabor served as the JBC's editor-in-chief. When he began his tenure, the fine structure and kinetics of only a few proteases were known; now thousands of proteases have been characterized, and over 600 genes for proteases have been identified in the human genome. In this review, besides reflecting on Dr. Tabor's invaluable contributions to the JBC and the American Society for Biochemistry and Molecular Biology (ASBMB), I endeavor to provide an overview of the extensive history of protease research, highlighting a few discoveries and roles of proteases in vivo . In addition, metalloproteinases, particularly meprins of the astacin family, will be discussed with regard to structural characteristics, regulation, mechanisms of action, and roles in health and disease. Proteases and protein degradation play crucial roles in living…

Monocarboxylate transporter 4 (MCT4) is an H + -coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30–40 mm, the reported K m of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective K m values for lactate uptake were 1.7, 1.2, and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a K m for pyruvate of 4.2 mm, as opposed to >150 mm reported previously. Parallel assays with the pH-sensitive dye 2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous K m…

Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-α kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy-related 13 (ATG13), Unc-51-like autophagy-activating kinases 1/2…

Renal ischemia/reperfusion injury (IRI) is a significant challenge in perioperative medicine and is related to oxidative programmed cell death. However, the role of ferroptosis, a newly discovered form of oxidative cell death, has not been evaluated widely. Pannexin 1 (PANX1), an ATP-releasing pathway family protein, has pro-apoptotic effects during kidney injury. Here, we demonstrate that PANX1 deletion protects against renal IRI by regulating ferroptotic cell death. Panx1 knockout mice subjected to renal IRI had decreased plasma creatinine, malondialdehyde (MDA) levels in kidney tissues, and tubular cell death (visible as decreased TUNEL-positive renal tubular cells) compared with WT mice. In cultured human kidney 2 (HK-2) cells, silenced Panx1 expression significantly attenuated ferroptotic lipid peroxidation and iron accumulation induced by the ferroptosis inducer erastin. Moreover, the Panx1 silencing significantly modulated ferroptosis-related protein expression. Furthermore, Panx1 deletion induced the expression of a cytoprotective chaperone, heme oxygenase-1 (HO-1), and…

Glycoside hydrolase family (GH) 16 comprises a large and taxonomically diverse family of glycosidases and transglycosidases that adopt a common ␤-jelly-roll fold and are active on a range of terrestrial and marine polysaccharides.Presently, broadly insightful sequence-function correlations in GH16 are hindered by a lack of a systematic subfamily structure.To fill this gap, we have used a highly scalable protein sequence similarity network analysis to delineate nearly 23,000 GH16 sequences into 23 robust subfamilies, which are strongly supported by hidden Markov model and maximum likelihood molecular phylogenetic analyses.Subsequent evaluation of over 40 experimental three-dimensional structures has highlighted key tertiary structural differences, predominantly manifested in active-site loops, that dictate substrate specificity across the GH16 evolutionary landscape.As for other large GH families (i.e.GH5, GH13, and GH43), this new subfamily classification provides a roadmap for functional glycogenomics that will guide future bioinformatics and experimental structurefunction…

The free radical nitric oxide (NO ⅐ ) exerts biological effects through the direct and reversible interaction with specific targets (e.g.soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules.The NO ⅐ -derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects.Their detection and quantification are technically challenging.In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species.In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NO ⅐ , and we discuss the approaches currently available for the analysis of NO ⅐ , nitrogen dioxide (NO 2 ⅐ ), dinitrogen trioxide (N 2 O 3 ), nitroxyl (HNO), and peroxynitrite (ONOO ؊ /ONOOH), as well as peroxynitrite-derived hydroxyl (HO ⅐ ) and carbonate anion (CO 3 ⅐ ؊ ) radicals.We also discuss the…

Similar to many other biological molecules, RNA is vulnerable to chemical insults from endogenous and exogenous sources. Noxious agents such as reactive oxygen species or alkylating chemicals have the potential to profoundly affect the chemical properties and hence the function of RNA molecules in the cell. Given the central role of RNA in many fundamental biological processes, including translation and splicing, changes to its chemical composition can have a detrimental impact on cellular fitness, with some evidence suggesting that RNA damage has roles in diseases such as neurodegenerative disorders. We are only just beginning to learn about how cells cope with RNA damage, with recent studies revealing the existence of quality-control processes that are capable of recognizing and degrading or repairing damaged RNA. Here, we begin by reviewing the most abundant types of chemical damage to RNA, including oxidation and alkylation. Focusing on mRNA damage, we then discuss how alterations to this species of RNA affect its function and how cells respond to these challenges to maintain…

Abstract Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however its role in the heart is poorly understood. Cardiac fibroblasts are responsible for preserving the structural integrity of the myocardium and play a key role in regulating its repair and remodeling following stress or injury. We investigated expression and function of Piezo1 in cultured human and mouse cardiac fibroblasts. RT-PCR studies confirmed expression of Piezo1 mRNA in cardiac fibroblasts at similar levels to endothelial cells. Fura-2 intracellular Ca 2+ measurements validated Piezo1 as a functional ion channel that was activated by the Piezo1 agonist, Yoda1. Yoda1-induced Ca 2+ entry was inhibited by Piezo1 blockers (gadolinium, ruthenium red) and the Ca 2+ response was reduced proportionally by Piezo1 siRNA knockdown or in cells from Piezo1 +/− mice. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation opposed cardiac fibroblast differentiation; data confirmed by functional collagen gel contraction assays. Piezo1 activation using…

Phase separation creates two distinct liquid phases from a single mixed liquid phase, like oil droplets separating from water. Considerable attention has focused on how the products of phase separation-the resulting condensates-might act as biological compartments, bioreactors, filters, and membraneless organelles in cells. Here, we expand this perspective, reviewing recent results showing how cells instead use the process of phase separation to sense intracellular and extracellular changes. We review case studies in phase separation-based sensing and discuss key features, such as extraordinary sensitivity, which make the process of phase separation ideally suited to meet a range of sensory challenges cells encounter.

G protein-coupled receptors (GPCRs) are the largest gene family of cell membrane-associated molecules mediating signal transmission, and their involvement in key physiological functions is well-established.The ability of GPCRs to regulate a vast array of fundamental biological processes, such as cardiovascular functions, immune responses, hormone and enzyme release from endocrine and exocrine glands, neurotransmission, and sensory perception (e.g.vision, odor, and taste), is largely due to the diversity of these receptors and the layers of their downstream signaling circuits.Dysregulated expression and aberrant functions of GPCRs have been linked to some of the most prevalent human diseases, which renders GPCRs one of the top targets for pharmaceutical drug development.However, the study of the role of GPCRs in tumor biology has only just begun to make headway.Recent studies have shown that GPCRs can contribute to the many facets of tumorigenesis, including proliferation, survival, angiogenesis, invasion, metastasis, therapy resistance, and immune evasion.Indeed, GPCRs are widely…

ABSTRACT Overexpressed Angiogenin (ANG) cleaves tRNA anticodons to produce tRNA halves similar to those produced in response to stress, but it is not clear whether endogenous ANG is essential for producing the stress-induced tRNA halves. It is also not clear whether smaller tRNA fragments (tRFs) are generated from the tRNA halves. Global short RNAseq experiments reveal that ANG over-expression selectively cleaves a subset of tRNAs (tRNA Glu , tRNA Gly , tRNA Lys , tRNA Val , tRNA His , tRNA Asp and tRNA SeC ) to produce tRNA halves and 26-30 bases long tRF-5s. Surprisingly, knockout of ANG reveals that the majority of stress-induced tRNA halves except 5’ half from tRNA HisGTG and 3’ half from tRNA AspGTC are ANG-independent, suggesting there are other RNases that can produce tRNA halves. The 17-25 bases long tRF-3s and tRF-5s that could enter into Argonaute complexes are not induced by ANG overexpression, suggesting that they are generated independently from tRNA halves. Consistent with this, knockout of ANG did not decrease tRF-3 levels or gene-silencing activity. Therefore ANG…

as a member of the Editorial Board beginning in 1961, as an Associate Editor, and as Editor-in-Chief for 40 years, from 1971 until 2010. Among the many discoveries in biological chemistry during this period was the identification of RNA modification by C6 deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA. This posttranscriptional RNA modification by adenosine deamination, known as A-to-I RNA editing, diversifies the transcriptome and modulates the innate immune interferon response. A-to-I editing is catalyzed by a family of enzymes, adenosine deaminases acting on dsRNA (ADARs). The roles of A-to-I editing are varied and include effects on mRNA translation, pre-mRNA splicing, and micro-RNA silencing. Suppression of dsRNA-triggered induction and action of interferon, the cornerstone of innate immunity, has emerged as a key function of ADAR1 editing of self (cellular) and nonself (viral) dsRNAs. A-to-I modification of RNA is essential for the normal regulation of cellular processes. Dysregulation of A-to-I editing by ADAR1 can have profound consequences,…

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely…

The ability of amyloid-β peptide (Aβ) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aβ is reported to have various impacts on the lipid bilayer, but a clearer picture of Aβ influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aβ assembly types on lipid bilayers. We show that only oligomeric Aβ can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aβ oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aβ monomers and fibers, although Aβ fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aβ oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1) G93A , revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1 G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain-…

Chimeric antigen receptor T-cell (CAR T-cell) therapy has been shown to be clinically effective for managing a variety of hematological cancers. However, CAR T-cell therapy is associated with multiple adverse effects, including neurotoxicity and cytokine release syndrome (CRS). CRS arises from massive cytokine secretion and can be life-threatening, but it is typically managed with an anti-IL-6Ra mAb or glucocorticoid administration. However, these treatments add to a patient's medication burden and address only the CRS symptoms. Therefore, alternative strategies that can prevent CRS and neurotoxicity associated with CAR T-cell treatment are urgently needed. Here, we explored a therapeutic route aimed at preventing CRS rather than limiting its consequences. Using a cytokine-profiling assay, we show that granulocyte–macrophage colony-stimulating factor (GMCSF) is a key CRS-promoting protein. Through a combination of in vitro experiments and gene-editing technology, we further demonstrate that antibody-mediated neutralization or TALEN-mediated genetic inactivation of GMCSF in CAR…

The catalytic residues of an enzyme comprise the amino acids located in the active center responsible for accelerating the enzyme-catalyzed reaction. These residues lower the activation energy of reactions by performing several catalytic functions. Decades of enzymology research has established general themes regarding the roles of specific residues in these catalytic reactions, but it has been more difficult to explore these roles in a more systematic way. Here, we review the data on the catalytic residues of 648 enzymes, as annotated in the Mechanism and Catalytic Site Atlas (M-CSA), and compare our results with those in previous studies. We structured this analysis around three key properties of the catalytic residues: amino acid type, catalytic function, and sequence conservation in homologous proteins. As expected, we observed that catalysis is mostly accomplished by a small set of residues performing a limited number of catalytic functions. Catalytic residues are typically highly conserved, but to a smaller degree in homologues that perform different reactions or are…

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in a lipopolysaccharide-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses lipopolysaccharide-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.

The ribosomally synthesized and posttranslationally modified peptides (RiPPs), also called ribosomal peptide natural products (RPNPs), form a growing superfamily of natural products that are produced by many different organisms and particularly by bacteria. They are derived from precursor polypeptides whose modification by various dedicated enzymes helps to establish a vast array of chemical motifs. RiPPs have attracted much interest as a source of potential therapeutic agents, and in particular as alternatives to conventional antibiotics to address the bacterial resistance crisis. However, their ecological roles in nature are poorly understood and explored. The present review describes major RiPP actors in competition within microbial communities, the main ecological and physiological functions currently evidenced for RiPPs, and the microbial ecosystems that are the sites for these functions. We envision that the study of RiPPs may lead to discoveries of new biological functions and highlight that a better knowledge of how bacterial RiPPs mediate inter-/intraspecies and…

Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and is evolutionarily conserved from yeast to humans. IRE1 possesses both Ser/Thr protein kinase and endoribonuclease (RNase) activities within its cytoplasmic domain and is activated through autophosphorylation and dimerization/oligomerization. It mediates a critical arm of the unfolded protein response to manage ER stress provoked by lumenal overload of unfolded/misfolded proteins. Emerging lines of evidence have revealed that in mammals, IRE1α functions as a multifunctional signal transducer that responds to metabolic cues and nutrient stress conditions, exerting profound and broad effects on metabolic homeostasis. In this review, we cover recent advances in our understanding of how IRE1α integrates a variety of metabolic and stress signals and highlight its tissue-specific or context-dependent metabolic activities. We also discuss how dysregulation of this metabolic stress sensor during handling of excessive nutrients in cells contributes to the progression of…

Protein disulfide bonds link pairs of cysteine residues in polypeptide chains. Many of these bonds serve a purely structural or energetic role, but a growing subset of cleavable disulfide bonds has been shown to control the function of the mature protein in which they reside. These allosteric disulfides and the factors that cleave these bonds are being identified across biological systems and life forms and have been shown to control hemostasis, the immune response, and viral infection in mammals. The discovery of these functional disulfides and a rationale for their facile nature has been aided by the emergence of a conformational signature for allosteric bonds. This post-translational modification mostly occurs extracellularly, making these chemical events prime drug targets. Indeed, a membrane-impermeable inhibitor of one of the cleaving factors is currently being trialed as an antithrombotic agent in cancer patients. Allosteric disulfides are firmly established as a sophisticated means by which a protein's shape and function can be altered; however, the full scope of this…

Fungi of the genus Trichoderma are a rich source of enzymes, such as cellulases and hemicellulases, that can degrade lignocellulosic biomass and are therefore of interest for biotechnological approaches seeking to optimize biofuel production. The essential transcription factor ACE3 is involved in cellulase production in Trichoderma reesei ; however, the mechanism by which ACE3 regulates cellulase activities is unknown. Here, we discovered that the nominal ace3 sequence in the T. reesei genome available through the Joint Genome Institute is erroneously annotated. Moreover, we identified the complete ace3 sequence, the ACE3 Zn(II) 2 Cys 6 domain, and the ACE3 DNA-binding sites containing a 5′-CGGAN(T/A) 3 -3′ consensus. We found that in addition to its essential role in cellulase production, ace3 is required for lactose assimilation and metabolism in T. reesei . Transcriptional profiling with RNA-Seq revealed that ace3 deletion down-regulates not only the bulk of the major cellulase, hemicellulase, and related transcription factor genes, but also reduces the expression of lactose…

N 6 -Methyladenosine (m 6 A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m 6 A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m 6 A sites on mRNAs. FMRP depletion increased mRNA m 6 A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1 -KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m 6 A-modified RNA targets.

Unlike most other tissues, the colon epithelium is exposed to high levels of H 2 S derived from gut microbial metabolism. H 2 S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H 2 S exposure as they harbor an efficient mitochondrial H 2 S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host–microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H 2 S poisoning. H 2 S at concentrations ≤20 μm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H 2 S concentrations as low as 5 μm poisoned…

to have the ability to "decide" whether to remain quiescent or to undergo activation upon signaling cues to support tissue function and homeostasis). Mitochondrial plasticity is thought to enable this reshaping of stem cell functions, integrating signaling cues with stem cell outcomes. Indeed, recent evidence highlights the crucial role of maintaining mitochondrial plasticity for stem cell biology. For example, tricarboxylic acid (TCA) cycle metabolites generated and metabolized in the mitochondria serve as cofactors for epigenetic enzymes, thereby coupling mitochondrial metabolism and transcriptional regulation. Another layer of mitochondrial plasticity has emerged, pointing toward mitochondrial dynamics in regulating stem cell fate decisions. Imposing imbalanced mitochondrial dynamics by manipulating the expression levels of the key molecular regulators of this process influences cellular outcomes by changing the nuclear transcriptional program. Moreover, reactive oxygen species have also been shown to play an important role in regulating transcriptional profiles in stem cells. In…

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of…

Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: the electron transfer chain (ETC), fatty acid β-oxidation (FAO), and the tricarboxylic acid cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano-LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings…

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-β production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I–induced IFN-β production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I–induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate–derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these…

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of ∼2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number T = 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a T = 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between…

Coronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all of the other structural proteins. Coronaviruses bud into the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC), but the mechanisms by which M proteins are transported from their site of synthesis, the ER, to the budding site remain poorly understood. Here, we investigated the intracellular trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M protein. Subcellular localization analyses revealed that the MERS-CoV M protein is retained intracellularly in the trans -Golgi network (TGN), and we identified two motifs in the distal part of the C-terminal domain as being important for this specific localization. We identified the first motif as a functional diacidic DxE ER export signal, because substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, 199 KxGxYR 204 , was responsible for retaining the M protein in the TGN. Substitution of this…

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated ␣-synuclein (␣-syn).These inclusions are associated with behavioral and pathological PD phenotypes.One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression.␣-Synuclein exists in multiple structural forms, including disordered, nonamyloid oligomers, ordered amyloid oligomers, and fibrils.It is critical to understand which conformers contribute to specific PD phenotypes.Here, we utilized a mouse model to explore the pathological effects of stable ␤-amyloid-sheet oligomers compared with those of fibrillar ␣-synuclein.We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays.We then injected these different ␣-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes.We found that ␤-sheet oligomers produce a small but significant loss of…

G protein-coupled receptors (GPCRs) play critical roles in regulating processes such as cellular homeostasis, responses to stimuli, and cell signaling. Accordingly, GPCRs have long served as extraordinarily successful drug targets. It is therefore not surprising that the discovery in the mid-1990s of a family of proteins that regulate processes downstream of GPCRs generated great excitement in the field. This finding enhanced the understanding of these critical signaling pathways and provided potentially new targets for pharmacological intervention. These regulators of G-protein signaling (RGS) proteins were viewed by many as nodes downstream of GPCRs that could be targeted with small molecules to tune signaling processes. In this review, we provide a brief overview of the discovery of RGS proteins and of the gradual and continuing discovery of their roles in disease states, focusing particularly on cancer and neurological disorders. We also discuss high-throughput screening efforts that have led to the discovery first of peptide-based and then of small-molecule inhibitors targeting…

In eukaryotes, the separation of translation from transcription by the nuclear envelope enables mRNA modifications such as capping, splicing, and polyadenylation. These modifications are mediated by a spectrum of ribonuclear proteins that associate with preRNA transcripts, coordinating the different steps and coupling them to nuclear export, ensuring that only mature transcripts reach the cytoplasmic translation machinery. Although the components of this machinery have been identified and considerable functional insight has been achieved, a number of questions remain outstanding about mRNA nuclear export and how it is integrated into the nuclear phase of the gene expression pathway. Nuclear export factors mediate mRNA transit through nuclear pores to the cytoplasm, after which these factors are removed from the mRNA, preventing transcripts from returning to the nucleus. However, as outlined in this review, several aspects of the mechanism by which transport factor binding and release are mediated remain unclear, as are the roles of accessory nuclear components in these processes.…

Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the…

Dietary, fructose-containing sugars have been strongly associated with the development of nonalcoholic fatty liver disease (NAFLD). Recent studies suggest that fructose also can be produced via the polyol pathway in the liver, where it may induce hepatic fat accumulation. Moreover, fructose metabolism yields uric acid, which is highly associated with NAFLD. Here, using biochemical assays, reporter gene expression, and confocal fluorescence microscopy, we investigated whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats and whether this stimulation is associated with endogenous fructose production and fat accumulation. Uric acid dose-dependently stimulated aldose reductase expression in the HepG2 cells, and this stimulation was associated with endogenous fructose production and triglyceride accumulation. This stimulatory mechanism was mediated by uric acid-induced oxidative stress and stimulation of…