The 50 most-cited articles in Journal of Biological Chemistry, 2018

Eukaryotic cells organize their intracellular components into organelles that can be membrane-bound or membraneless. A large number of membraneless organelles, including nucleoli, Cajal bodies, P-bodies, and stress granules, exist as liquid droplets within the cell and arise from the condensation of cellular material in a process termed liquid-liquid phase separation (LLPS). Beyond a mere organizational tool, concentrating cellular components into membraneless organelles tunes biochemical reactions and improves cellular fitness during stress. In this review, we provide an overview of the molecular underpinnings of the formation and regulation of these membraneless organelles. This molecular understanding explains emergent properties of these membraneless organelles and shines new light on neurodegenerative diseases, which may originate from disturbances in LLPS and membraneless organelles.

Yersinia pseudotuberculosis is a Gram-negative bacterium and zoonotic pathogen responsible for a wide range of diseases, ranging from mild diarrhea, enterocolitis, lymphatic adenitis to persistent local inflammation.The Y. pseudotuberculosis invasin D (InvD) molecule belongs to the invasin (InvA)-type autotransporter proteins, but its structure and function remain unknown.In this study, we present the first crystal structure of InvD, analyzed its expression and function in a murine infection model, and identified its target molecule in the host.We found that InvD is induced at 37 °C and expressed in vivo 2-4 days after infection, indicating that InvD is a virulence factor.During infection, InvD was expressed in all parts of the intestinal tract, but not in deeper lymphoid tissues.The crystal structure of the C-terminal adhesion domain of InvD revealed a distinct Ig-related fold that, apart from the canonical ␤-sheets, comprises various modifications of and insertions into the Ig-core structure.We identified the Fab fragment of host-derived IgG/IgA antibodies as the target of the…

, particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes. Aspects of DNA topology affecting individual steps are highlighted. Overall, recombination is a dynamic pathway with multiple metastable and reversible intermediates designed to achieve DNA repair with high fidelity.

The endoplasmic reticulum (ER) represents the entry point into the secretory pathway where nascent proteins encounter a specialized environment for their folding and maturation. Inherent to these processes is a dedicated quality-control system that detects proteins that fail to mature properly and targets them for cytosolic degradation. An imbalance in protein folding and degradation can result in the accumulation of unfolded proteins in the ER, resulting in the activation of a signaling cascade that restores proper homeostasis in this organelle. The ER heat shock protein 70 (Hsp70) family member BiP is an ATP-dependent chaperone that plays a critical role in these processes. BiP interacts with specific ER-localized DnaJ family members (ERdjs), which stimulate BiP's ATP-dependent substrate interactions, with several ERdjs also binding directly to unfolded protein clients. Recent structural and biochemical studies have provided detailed insights into the allosteric regulation of client binding by BiP and have enhanced our understanding of how specific ERdjs enable BiP to perform its…

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing…

Ribonucleoprotein (RNP) granules are membrane-less organelles consisting of RNA-binding proteins (RBPs) and RNA. RNA granules form through liquid-liquid phase separation (LLPS), whereby weak promiscuous interactions among RBPs and/or RNAs create a dense network of interacting macromolecules and drive the phase separation. Post-translational modifications (PTMs) of RBPs have emerged as important regulators of LLPS and RNP granule dynamics, as they can directly weaken or enhance the multivalent interactions between phase-separating macromolecules or can recruit or exclude certain macromolecules into or from condensates. Here, we review recent insights into how PTMs regulate phase separation and RNP granule dynamics, in particular arginine (Arg)-methylation and phosphorylation. We discuss how these PTMs regulate the phase behavior of prototypical RBPs and how, as "friend or foe," they might influence the assembly, disassembly, or material properties of cellular RNP granules, such as stress granules or amyloid-like condensates. We particularly highlight how PTMs control the phase…

and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide the groundwork for understanding the more complex eukaryotic Hsp90 system and its modulation by Hsp90 co-chaperones.

Abstract Polycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and…

production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.

DNA double-strand breaks (DSBs) arise regularly in cells and when left unrepaired cause senescence or cell death. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two major DNA-repair pathways. Whereas HR allows faithful DSB repair and healthy cell growth, NHEJ has higher potential to contribute to mutations and malignancy. Many regulatory mechanisms influence which of these two pathways is used in DSB repair. These mechanisms depend on the cell cycle, post-translational modifications, and chromatin effects. Here, we summarize current research into these mechanisms, with a focus on mammalian cells, and also discuss repair by "alternative end-joining" and single-strand annealing.

Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide–dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H 2 O 2 ) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring…

Hsp70 chaperones are central hubs of the protein quality control network and collaborate with co-chaperones having a J-domain (an ∼70-residue-long helical hairpin with a flexible loop and a conserved His-Pro-Asp motif required for ATP hydrolysis by Hsp70s) and also with nucleotide exchange factors to facilitate many protein-folding processes that (re)establish protein homeostasis. The Hsp70s are highly dynamic nanomachines that modulate the conformation of their substrate polypeptides by transiently binding to short, mostly hydrophobic stretches. This interaction is regulated by an intricate allosteric mechanism. The J-domain co-chaperones target Hsp70 to their polypeptide substrates, and the nucleotide exchange factors regulate the lifetime of the Hsp70-substrate complexes. Significant advances in recent years are beginning to unravel the molecular mechanism of this chaperone machine and how they treat their substrate proteins.

Small heat shock proteins (sHsps) are a ubiquitous and ancient family of ATP-independent molecular chaperones. A key characteristic of sHsps is that they exist in ensembles of iso-energetic oligomeric species differing in size. This property arises from a unique mode of assembly involving several parts of the subunits in a flexible manner. Current evidence suggests that smaller oligomers are more active chaperones. Thus, a shift in the equilibrium of the sHsp ensemble allows regulating the chaperone activity. Different mechanisms have been identified that reversibly change the oligomer equilibrium. The promiscuous interaction with non-native proteins generates complexes that can form aggregate-like structures from which native proteins are restored by ATP-dependent chaperones such as Hsp70 family members. In recent years, this basic paradigm has been expanded, and new roles and new cofactors, as well as variations in structure and regulation of sHsps, have emerged.

They also first purified the extracellular bovine serum amine oxidase and elucidated the products of its oxidation of primary amine groups of PAs when included in culture medium. These investigations formed the foundation for many polyamine-related studies and experimental procedures still performed today. This Minireview will summarize key innovative studies regarding PAs and oxidative damage, starting with those from the Tabor laboratory and including the most recent advances, with a focus on mammalian systems.

Eukaryotic cells contain distinct organelles, but not all of these compartments are enclosed by membranes. Some intrinsically disordered proteins mediate membraneless organelle formation through liquid-liquid phase separation (LLPS). LLPS facilitates many biological functions such as regulating RNA stability and ribonucleoprotein assembly, and disruption of LLPS pathways has been implicated in several diseases. Proteins exhibiting LLPS typically have low sequence complexity and specific repeat motifs. These motifs promote multivalent connections with other molecules and the formation of higher-order oligomers, and their removal usually prevents LLPS. The intrinsically disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43), a protein involved in motor neuron disease and dementia lacks a dominant LLPS motif, however, and how this domain forms condensates is unclear. Using extensive mutagenesis of TDP-43, we demonstrate here that three tryptophan residues and, to a lesser extent, four other aromatic residues are most important for TDP-43 to undergo LLPS. Our results also…

Autophagy is a highly conserved process and is essential for the maintenance of cellular homeostasis. Autophagy occurs at a basal level in all cells, but it can be up-regulated during stress, starvation, or infection. Misregulation of autophagy has been linked to various disorders, including cancer, neurodegeneration, and immune diseases. Here, we discuss the essential proteins acting in the formation of an autophagosome, with a focus on the ULK and VPS34 kinase complexes, phosphatidylinositol 3-phosphate effector proteins, and the transmembrane autophagy-related protein ATG9. The function and regulation of these and other autophagy-related proteins acting during formation will be addressed, in particular during amino acid starvation.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout ( LDHA/B -DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B -DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory…

The Hippo pathway plays an important role in regulating tissue homeostasis, and its effectors, the transcriptional co-activators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1 or TAZ), are responsible for mediating the vast majority of its physiological functions. Although YAP and TAZ are thought to be largely redundant and similarly regulated by Hippo signaling, they have developmental, structural, and physiological differences that suggest they may differ in their regulation and downstream functions. To better understand the functions of YAP and TAZ in the Hippo pathway, using CRISPR/Cas9, we generated YAP KO, TAZ KO, and YAP/TAZ KO cell lines in HEK293A cells. We evaluated them in response to many environmental conditions and stimuli and used RNA-Seq to compare their transcriptional profiles. We found that YAP inactivation has a greater effect on cellular physiology (namely, cell spreading, volume, granularity, glucose uptake, proliferation, and migration) than TAZ inactivation. However, functional redundancy between YAP and TAZ was also…

The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1β. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and…

Pioneer transcription factors have the unique and important role of unmasking chromatin domains during development to allow the implementation of new cellular programs. Compared with those of other transcription factors, this activity implies that pioneer factors can recognize their target DNA sequences in so-called compacted or "closed" heterochromatin and can trigger remodeling of the adjoining chromatin landscape to provide accessibility to nonpioneer transcription factors. Recent studies identified several steps of pioneer action, namely rapid but weak initial binding to heterochromatin and stabilization of binding followed by chromatin opening and loss of cytosine-phosphate-guanine (CpG) methylation that provides epigenetic memory. Whereas CpG demethylation depends on replication, chromatin opening does not. In this Minireview, we highlight the unique properties of this transcription factor class and the challenges of understanding their mechanism of action.

Interleukin (IL-)6 is the major pro-inflammatory cytokine within the IL-6 family. IL-6 signals via glycoprotein 130 (gp130) and the membrane-bound or soluble IL-6 receptor (IL-6R), referred to as classic or trans-signaling, respectively. Whereas inflammation triggers IL-6 expression, eventually rising to nanogram/ml serum levels, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) are constitutively present in the upper nanogram/ml range. Calculations based on intermolecular affinities have suggested that systemic IL-6 is immediately trapped in IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system that increases the serum half-life of IL-6 or restricts systemic IL-6 signaling. However, this scenario has not been experimentally validated. Here, we quantified IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes over a wide concentration range. The amounts of IL-6 used in this study reflect concentrations found during active inflammatory events. Our results indicated that most IL-6 is free and not complexed with sIL-6R or sgp130, indicating…

The mammalian SWI/SNF chromatin remodeling complex is a heterogeneous collection of related protein complexes required for gene regulation and genome integrity. It contains a central ATPase (BRM or BRG1) and various combinations of 10–14 accessory subunits (BAFs for BRM/BRG1 Associated Factors). Two distinct complexes differing in size, BAF and the slightly larger polybromo-BAF (PBAF), share many of the same core subunits but are differentiated primarily by having either AT-rich interaction domain 1A/B (ARID1A/B in BAF) or ARID2 (in PBAF). Using density gradient centrifugation and immunoprecipitation, we have identified and characterized a third and smaller SWI/SNF subcomplex. We termed this complex GBAF because it incorporates two mutually exclusive paralogs, GLTSCR1 (glioma tumor suppressor candidate region gene 1) or GLTSCR1L (GLTSCR1-like), instead of an ARID protein. In addition to GLTSCR1 or GLTSCR1L, the GBAF complex contains BRD9 (bromodomain-containing 9) and the BAF subunits BAF155, BAF60, SS18, BAF53a, and BRG1/BRM. We observed that GBAF does not contain the core BAF…

Alternative end-joining (a-EJ) pathways, which repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3' single strands. This reaction is shared, at least in part, with homologous recombination but distinguishes a-EJ from the major nonhomologous end-joining pathway. Although the a-EJ pathways make only a minor and poorly understood contribution to DSB repair in nonmalignant cells, there is growing interest in these pathways, as they generate genomic rearrangements that are hallmarks of cancer cells. Here, we review and discuss the current understanding of the mechanisms and regulation of a-EJ pathways, the role of a-EJ in human disease, and the potential utility of a-EJ as a therapeutic target in cancer.

Membrane biology seeks to understand how lipids and proteins within bilayers assemble into large structures such as organelles and the plasma membranes. Historically, lipids were thought to merely provide structural support for bilayer formation and membrane protein function. Research has now revealed that phospholipid metabolism regulates nearly all cellular processes. Sophisticated techniques helped identify >10,000 lipid species suggesting that lipids support many biological processes. Here, we highlight the synthesis of the most abundant glycerophospholipid classes and their distribution in organelles. We review vesicular and nonvesicular transport pathways shuttling lipids between organelles and discuss lipid regulators of membrane trafficking and second messengers in eukaryotic cells.

Many proteins, such as RNA-binding proteins, have complex folding landscapes. How cells maintain the solubility and folding state of such proteins, particularly under stress conditions, is largely unknown. Here, we argue that prion-like low-complexity regions (LCRs) are key regulators of protein solubility and folding. We discuss emerging evidence that prion-like LCRs are not, as commonly thought, autonomous aggregation modules that adopt amyloid-like conformations, but protein-specific sequences with chaperone-like functions. On the basis of recent findings, we propose that prion-like LCRs have evolved to regulate protein phase behavior and to protect proteins against proteotoxic damage.

More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer Ostrinia furnacalis , we identified a gene, termed up56 , that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that up56 encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in O. furnacalis strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both in vitro and in vivo . Of note, we found that this…

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant…

Parkinson's disease (PD) and multiple system atrophy (MSA) are distinct clinical syndromes characterized by the pathological accumulation of α-synuclein (α-syn) protein fibrils in neurons and glial cells. These disorders and other neurodegenerative diseases may progress via prion-like mechanisms. The prion model of propagation predicts the existence of "strains" that link pathological aggregate structure and neuropathology. Prion strains are aggregated conformers that stably propagate in vivo and cause disease with defined incubation times and patterns of neuropathology. Indeed, tau prions have been well defined, and research suggests that both α-syn and β-amyloid may also form strains. However, there is a lack of studies characterizing PD- versus MSA-derived α-syn strains or demonstrating stable propagation of these unique conformers between cells or animals. To fill this gap, we used an assay based on FRET that exploits a HEK293T "biosensor" cell line stably expressing α-syn (A53T)-CFP/YFP fusion proteins to detect α-syn seeds in brain extracts from PD and MSA patients. Both…

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD's anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC 50 of 1.9–3.8 μm, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade,…

-4-amino-2-hydroxybutyl(lysine)) is derived from the polyamine spermidine, and occurs only in a single family of cellular proteins, eukaryotic translation factor 5A (eIF5A) isoforms. Hypusine is formed by conjugation of the aminobutyl moiety of spermidine to a specific lysine residue of this protein. The posttranslational synthesis of hypusine involves two enzymatic steps, catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusine is essential for eIF5A activity. Inactivation of either the eIF5A or the DHPS gene is lethal in yeast and mouse, underscoring the vital role of eIF5A hypusination in eukaryotic cell growth and animal development. The long and basic side chain of the hypusine residue promotes eIF5A-mediated translation elongation by facilitating peptide bond formation at polyproline stretches and at many other ribosome-pausing sites. It also enhances translation termination by stimulating peptide release. By promoting translation, the hypusine modification of eIF5A provides a key link between polyamines and cell growth regulation. eIF5A has…

As a central hub of cellular metabolism and signaling, the mitochondrion is a crucial organelle whose dysfunction can cause disease and whose activity is intimately connected to aging. We review how the mitochondrial network maintains proteomic integrity, how mitochondrial proteotoxic stress is communicated and resolved in the context of the entire cell, and how mitochondrial systems function in the context of organismal health and aging. A deeper understanding of how mitochondrial protein quality control mechanisms are coordinated across these distinct biological levels should help explain why these mechanisms fail with age and, ultimately, how routes to intervention might be attained.

Most of the phylogenetic diversity of life is found in bacteria and archaea, and is reflected in the diverse metabolism and functions of bacterial and archaeal polyamines. The polyamine spermidine was probably present in the last universal common ancestor, and polyamines are known to be necessary for critical physiological functions in bacteria, such as growth, biofilm formation, and other surface behaviors, and production of natural products, such as siderophores. There is also phylogenetic diversity of function, indicated by the role of polyamines in planktonic growth of different species, ranging from absolutely essential to entirely dispensable. However, the cellular molecular mechanisms responsible for polyamine function in bacterial growth are almost entirely unknown. In contrast, the molecular mechanisms of essential polyamine functions in archaea are better understood: covalent modification by polyamines of translation factor aIF5A and the agmatine modification of tRNA Ile . As with bacterial hyperthermophiles, archaeal thermophiles require long-chain and branched polyamines…

Coronavirus tropism is predominantly determined by the interaction between coronavirus spikes and the host receptors. In this regard, coronaviruses have evolved a complicated receptor-recognition system through their spike proteins. Spikes from highly related coronaviruses can recognize distinct receptors, whereas spikes of distant coronaviruses can employ the same cell-surface molecule for entry. Moreover, coronavirus spikes can recognize a broad range of cell-surface molecules in addition to the receptors and thereby can augment coronavirus attachment or entry. The receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) is dipeptidyl peptidase 4 (DPP4). In this study, we identified membrane-associated 78-kDa glucose-regulated protein (GRP78) as an additional binding target of the MERS-CoV spike. Further analyses indicated that GRP78 could not independently render nonpermissive cells susceptible to MERS-CoV infection but could facilitate MERS-CoV entry into permissive cells by augmenting virus attachment. More importantly, by exploring potential interactions between…

Glycogen, the primary storage form of glucose, is a rapid and accessible form of energy that can be supplied to tissues on demand. Each glycogen granule, or "glycosome," is considered an independent metabolic unit composed of a highly branched polysaccharide and various proteins involved in its metabolism. In this Minireview, we review the literature to follow the dynamic life of a glycogen granule in a multicompartmentalized system, i.e. the cell, and how and where glycogen granules appear and the factors governing its degradation. A better understanding of the importance of cellular compartmentalization as a regulator of glycogen metabolism is needed to unravel its role in brain energetics.

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes…

Abstract Transcellular propagation of aggregate “seeds” has been proposed to mediate progression of neurodegenerative diseases in tauopathies and α-synucleinopathies. We have previously determined that tau and α-synuclein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface. This mediates uptake and intracellular seeding. The specificity and mode of binding to HSPGs has been unknown. We used modified heparins to determine the size and sulfation requirements of glycosaminoglycan (GAGs) binding to aggregates in biochemical and cell uptake and seeding assays. Aggregates of tau require a precise GAG architecture with defined sulfate moieties in the N- and 6-O-positions, whereas α-synuclein and Aβ rely slightly more on overall charge on the GAGs. To determine the genetic requirements for aggregate uptake, we individually knocked out the major genes of the HSPG synthesis pathway using CRISPR/Cas9 in HEK293T cells. Knockout of EXT1, EXT2 and EXTL3, N-sulfotransferase (NDST1), and 6-O-sulfotransferase (HS6ST2) significantly reduced tau uptake. α-Synuclein was not…

Microglia are immune cells of the central nervous system and are implicated in brain inflammation. However, how brain microglia modulate transport and metabolism of the essential metal iron in response to pro- and anti-inflammatory environmental cues is unclear. Here, we characterized uptake of transferrin (Tf)-bound iron (TBI) and non-Tf-bound iron (NTBI) by immortalized microglial (IMG) cells. We found that these cells preferentially take up NTBI in response to the proinflammatory stimulus lipopolysaccharide (LPS) or β-amyloid (Aβ). In contrast, the anti-inflammatory cytokine interleukin 4 (IL-4) promoted TBI uptake. Concordant with these functional data, levels of the Tf receptor (TfR) in IMG cells were up-regulated in response to IL-4, whereas divalent metal transporter-1 (DMT1) and ferritin levels increased in response to LPS or Aβ. Similar changes in expression were confirmed in isolated primary adult mouse microglia treated with pro- or anti-inflammatory inducers. LPS-induced changes in IMG cell iron metabolism were accompanied by notable metabolic changes, including…

Polyphosphate (polyP) consists of a linear arrangement of inorganic phosphates and defies its structural simplicity with an astounding number of different activities in the cell. Already well known for its ability to partake in phosphate, calcium, and energy metabolism, polyP recently gained a new functional dimension with the discovery that it serves as a stabilizing scaffold for protein-folding intermediates. In this review, we summarize and discuss the recent studies that have identified polyP not only as a potent protein-like chaperone that protects cells against stress-induced protein aggregation, but also as a robust modifier of amyloidogenic processes that shields neuronal cells from amyloid toxicity. We consider some of the most pressing questions in the field, the obstacles faced, and the potential avenues to take to provide a complete picture about the working mechanism and physiological relevance of this intriguing biopolymer.

The transporters for glutamine and essential amino acids, ASCT2 (solute carrier family 1 member 5, SLC1A5) and LAT1 (solute carrier family 7 member 5, SLC7A5), respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry via the LAT1 exchanger, thus activating mechanistic target of rapamycin complex 1 (mTORC1) and stimulating growth. Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2 KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549- ASCT2 KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1 KO cells, ASCT2 KO cells displayed no…

Heat shock protein 47 (Hsp47) is an endoplasmic reticulum (ER)-resident molecular chaperone essential for correct folding of procollagen in mammalian cells. In this Review, we discuss the role and function of Hsp47 in vertebrate cells and its role in connective tissue disorders. Hsp47 binds to collagenous (Gly–Xaa–Arg) repeats within triple-helical procollagen in the ER and can prevent its local unfolding or aggregate formation, resulting in accelerating triple-helix formation of procollagen. Hsp47 pH-dependently dissociates from procollagen in the cis -Golgi or ER–Golgi intermediate compartment and is then transported back to the ER. Although Hsp47 belongs to the serine protease inhibitor (serpin) superfamily, it does not possess serine protease inhibitory activity. Whereas general molecular chaperones such as Hsp70 and Hsp90 exhibit broad substrate specificity, Hsp47 has narrower specificity mainly for procollagens. However, other Hsp47-interacting proteins have been recently reported, suggesting a much broader role for Hsp47 in the cell that warrants further investigation. Other…

When properly employed, targeted therapies are effective cancer treatments. However, the development of such therapies requires the identification of targetable drivers of cancer development and metastasis. The expression and nuclear localization of the transcriptional coactivators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are increased in many human cancers, and experimental evidence indicates that aberrant YAP or TAZ activation drives tumor formation and metastasis. Although these findings make YAP and TAZ appealing therapeutic targets, both have important functions in adult tissues, so directly targeting them could cause adverse effects. The identification of pathways active in cancer cells and required for YAP/TAZ activity could provide a way to inhibit YAP and TAZ. Here, we show that SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) is an important driver of YAP/TAZ activity in human breast cancer and melanoma cells. SRC activation increased YAP/TAZ activity and the expression of YAP/TAZ-regulated genes. In contrast, SRC…

Bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) can differentiate into osteoblasts (OBs), adipocytes, or chondrocytes. As BMSCs undergo OB differentiation, they up-regulate mitochondrial oxidative phosphorylation (OxPhos). Here, we investigated the mechanism(s) connecting mitochondrial OxPhos to OB differentiation. First, we found that treating BMSC-like C3H10T1/2 cells with an OxPhos inhibitor reduces their osteogenic potential. Interestingly, ATP levels were not reduced, as glycolysis compensated for the decreased OxPhos. Thus, mitochondria support OB differentiation not only by supplying ATP, but also by other mechanisms. To uncover these mechanisms, we stimulated OxPhos in C3H10T1/2 cells by replacing media glucose with galactose and observed that this substitution increases both OxPhos and osteogenesis even in the absence of osteoinducers. β-Catenin, an important signaling pathway in osteogenesis, was found to be responsive to OxPhos stimulation. β-Catenin activity is maintained by acetylation, and mitochondria generate the acetyl donor acetyl-CoA, which upon…

Adaptations and responses to stress conditions are fundamental processes that all cells must accomplish to maintain or restore cellular homeostasis. Cells have a plethora of response pathways to mitigate the effect of different environmental stressors. The transcriptional regulators transcription factor EB (TFEB) and transcription factor binding to IGHM enhancer 3 (TFE3) play a key role in the control of these stress pathways. Therefore, understanding their regulation under different stress conditions is of great interest. Here, using a range of human and murine cells, we show that TFEB and TFE3 are activated upon induction of acute oxidative stress by sodium arsenite via an mTOR complex 1 (mTORC1)-independent process. We found that the mechanism of arsenite-stimulated TFEB and TFE3 activation instead involves protein phosphatase 2A (PP2A)-mediated dephosphorylation at Ser-211 and Ser-321, respectively. Depletion of either the catalytic (PPP2CA+B) or regulatory (PPP2R2A/B55α) subunits of PP2A, as well as PP2A inactivation with the specific inhibitor okadaic acid, abolished TFEB and…

The aim of the current study was to investigate the effects of glucosamine (GlcN) on septic lethality and sepsis-induced inflammation using animal models of mice and zebrafish. GlcN pretreatment improved survival in the cecal ligation and puncture (CLP)-induced sepsis mouse model and attenuated lipopolysaccharide (LPS)-induced septic lung injury and systemic inflammation. GlcN suppressed LPS-induced M1-specific but not M2-specific gene expression. Furthermore, increased expressions of inflammatory genes in visceral tissue of LPS-injected zebrafish were suppressed by GlcN. GlcN suppressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and NF-κB in lung tissue. LPS triggered a reduction in O -GlcNAc levels in nucleocytoplasmic proteins of lung, liver, and spleen after 1 day, which returned to normal levels at day 3. GlcN inhibited LPS-induced O -GlcNAc down-regulation in mouse lung and visceral tissue of zebrafish. Furthermore, the O -GlcNAcase (OGA) level was increased by LPS, which were suppressed by GlcN in mouse and zebrafish. OGA inhibitors suppressed…

ABSTRACT Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous m etabolite, p rotein, and l ipid e xtraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and…

The brain contains a fairly low amount of glycogen, mostly located in astrocytes, a fact that has prompted the suggestion that glycogen does not have a significant physiological role in the brain. However, glycogen metabolism in astrocytes is essential for several key physiological processes and is adversely affected in disease. For instance, diminished ability to break down glycogen impinges on learning, and epilepsy, Alzheimer's disease, and type 2 diabetes are all associated with abnormal astrocyte glycogen metabolism. Glycogen metabolism supports astrocytic K + and neurotransmitter glutamate uptake and subsequent glutamine synthesis—three fundamental steps in excitatory signaling at most brain synapses. Thus, there is abundant evidence for a key role of glycogen in brain function. Here, we summarize the physiological brain functions that depend on glycogen, discuss glycogen metabolism in disease, and investigate how glycogen breakdown is regulated at the cellular and molecular levels.

Generating a functional proteome requires the ribosome to carefully regulate disparate co-translational processes that determine the fate of nascent polypeptides. With protein synthesis being energetically expensive, the ribosome must balance the costs of efficiently making a protein with those of properly folding it. Emerging as a primary means of regulating this trade-off is the nonuniform rate of translation elongation that defines translation kinetics. The varying speeds with which the ribosome progresses along a transcript have been implicated in several aspects of protein biogenesis, including co-translational protein folding and translational fidelity, as well as gene expression by mediating mRNA decay and protein quality control pathways. The optimal translation kinetics required to efficiently execute these processes can be distinct. Thus, the ribosome is tasked with tightly regulating translation kinetics to balance these processes while maintaining adaptability for changing cellular conditions. In this review, we first discuss the regulatory role of translation elongation…

Toll-like receptors (TLRs) form part of the host innate immune system, in which they act as sensors of microbial and endogenous danger signals. Upon TLR activation, the intracellular Toll/interleukin-1 receptor domains of TLR dimers initiate oligomerization of a multiprotein signaling platform comprising myeloid differentiation primary response 88 (MyD88) and members of the interleukin-1 receptor-associated kinase (IRAK) family. Formation of this myddosome complex initiates signal transduction pathways, leading to the activation of transcription factors and the production of inflammatory cytokines. To date, little is known about the assembly and disassembly of the myddosome and about the mechanisms by which these complexes mediate multiple downstream signaling pathways. Here, we isolated myddosome complexes from whole-cell lysates of TLR-activated primary mouse macrophages and from IRAK reporter macrophages to examine the kinetics of myddosome assembly and disassembly. Using a selective inhibitor of IRAK4's kinase activity, we found that whereas TLR cytokine responses were ablated,…

can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 μm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ…

Increased oxidative stress and abundance of reactive oxygen species (ROS) are positively correlated with a variety of pathophysiologies, including cardiovascular disease, type 2 diabetes, Alzheimer's disease, and neuroinflammation. In adipose biology, diabetic obesity is correlated with increased ROS in an age- and depot-specific manner and is mechanistically linked to mitochondrial dysfunction, endoplasmic reticulum (ER) stress, potentiated lipolysis, and insulin resistance. The cellular quality control systems that homeostatically regulate oxidative stress in the lean state are down-regulated in obesity as a consequence of inflammatory cytokine pressure leading to the accumulation of oxidized biomolecules. New findings have linked protein, DNA, and lipid oxidation at the biochemical level, and the structures and potential functions of protein adducts such as carbonylation that accumulate in stressed cells have been characterized. The sum total of such regulation and biochemical changes results in alteration of cellular metabolism and function in the obese state relative to the…