Biology subjects

Jaiswal, N.

Publications and source records attributed to Jaiswal, N..

18 recordsLinked to original sources

bioRxiv2026

Dietary omega-6 arachidonic acid and omega-3 docosahexaenoic acid supplementation differentially impact skeletal muscle inflammaging in mice

Aging is associated with a gradual and progressive decline in skeletal muscle mass and strength known as sarcopenia, which has been attributed to chronic low-grade inflammation. Dietary long-chain polyunsaturated fatty acids (LC-PUFAs), including omega-6 arachidonic acid (ARA) and omega-3 docosahexaenoic acid (DHA), are precursors to bioactive lipid mediators that regulate the initiation, propagation, and active resolution of inflammation. While traditionally considered a pro-inflammatory and catabolic factor, the ARA-derived eicosanoid prostaglandin E2 has recently emerged as a potential anti-sarcopenic molecule. DHA-derived specialized pro-resolving mediators may also act as immunomodulatory pro-regenerative molecules in muscle inflammaging. In the current study, we tested the effects of long-term dietary supplementation with either ARA or DHA on muscle health in aging mice. Twenty-two-month-old C57BL/6N mice were fed a control AIN-93M diet, or an AIN-93M diet supplemented with either ARA (0.48% w/w) or DHA (0.48% w/w) for 12 weeks. Both dietary interventions reduced total body weight, but only ARA reduced absolute fat mass and increased the percentage of lean mass. Despite these changes in body composition, ARA supplementation reduced absolute muscle strength and myofiber size. This functional decline was associated with increased neuromuscular junction fragmentation, elevated expression of pro-inflammatory cytokines/protein degradation markers, and suppressed ribosome biogenesis. In contrast, DHA uniquely reduced chronic inflammation of aged muscle and returned c-Myc expression to young levels but did not affect muscle mass or strength. These data demonstrate that long-term dietary intake of ARA and DHA have overall divergent effects on the structure and function of aging muscle.

physiology↗

bioRxiv2026

Lactylation landscape of mitochondrial proteins in myocardial infarction

Metabolic reprogramming is a hallmark of myocardial infarction (MI), in which cardiomyocytes shift from fatty acid oxidation to anaerobic glycolysis, leading to elevated lactate production and mitochondrial dysfunction. Lactylation, a recently described lysine post-translational modification, has emerged as a metabolic signaling mechanism; however, its role within mitochondria during MI remains poorly understood. Here, we define the mitochondrial lactylome following MI and examine how modulation of lactate transport influences mitochondrial metabolism and redox homeostasis. Using quantitative proteomics, we identify extensive remodeling of mitochondrial protein lactylation after MI, affecting enzymes involved in bioenergetics, redox regulation, and metabolic control. Pharmacological inhibition of monocarboxylate transporter-1 (MCT1) using AZD3965 further reshapes the mitochondrial lactylome, increasing lactylation of specific metabolic and redox-associated proteins without uniformly exacerbating mitochondrial dysfunction. Despite sustained impairment of global cardiac function, MCT1 inhibition attenuates post-MI fibrosis and inflammation and partially restores mitochondrial respiratory capacity. Consistent with in vivo findings, genetic or pharmacological inhibition of MCT1 in hypoxic cardiomyocytes-derived cells reduces mitochondrial reactive oxygen species, decreases inhibitory pyruvate dehydrogenase phosphorylation, and improves mitochondrial bioenergetics. Together, these findings reveal that mitochondrial lactylation is a context-dependent regulator of mitochondrial metabolism and redox balance following MI. Rather than acting solely as a pathological modification, lactylation integrates lactate availability with mitochondrial function to influence inflammatory and fibrotic remodeling, highlighting mitochondrial metabolic plasticity as a potential therapeutic target in ischemic heart disease. HighlightsO_LIMyocardial infarction (MI) increases mitochondrial protein lactylation, with 361 identified lactylated proteins. C_LIO_LIAZD3965-mediated MCT1 inhibition further elevates mitochondrial lactylation. C_LIO_LIDistinct alterations in mitochondrial proteins and pathways (TCA cycle, amino acid metabolism, gene expression) were observed. C_LIO_LIAZD3965 reduces cardiac fibrosis and inflammation and partly improves mitochondrial respiration post-MI, but cardiac function remains impaired. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/718938v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@b5a7b3org.highwire.dtl.DTLVardef@14ea92org.highwire.dtl.DTLVardef@1343a29org.highwire.dtl.DTLVardef@1d67716_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2026

''Self-gapping'' by a C-terminal domain arginine finger regulates GTP hydrolysis in bacterial zinc metallochaperones

The cellular response to transition metal scarcity is multifaceted and complex. Members of the Cluster of Orthologous Groups 0523 (COG0523) superfamily are proposed to chaperone a bound metal to activate an apoenzyme client and are thus candidate metallochaperones. COG0523 enzymes are GTPases that harbor a conserved Ras-like GTP-binding and hydrolysis domain (G-domain) and a C-terminal domain (CTD) of unknown function connected by a flexible linker. AlphaFold3 modeling posits an "open" GTPase-inactive and "closed" GTPase-active conformation where the GTP and switch 1 (G2) loop are buried at the interface of the two domains. We show here that the CTD functions as a GTP-hydrolysis activation protein (GAP) domain that stimulates GTP hydrolysis by the tethered G-domain. This "self-gapping" activity requires an invariant RxKG sequence in the {beta}2-strand of the CTD in two distantly related bacterial COG0523s from Acinetobacter baumannii, ZigA and MigC. Thermodynamic and kinetic studies reveal that the Arg is analogous to the "arginine finger" motif of a Ras-cognate GAP, while the Lys residue appears to play a catalytic role in GTP hydrolysis. Cognate CTD added in trans to full-length RxK mutant ZigA or MigC rescues Zn(II)-activated GTPase activity whereas the non-cognate CTD shows no rescue. The linker in AbZigA appears to gate Zn(II)-stimulated GTP hydrolysis. Solution NMR studies of RxK AbMigC reveal that the two domains tumble independently of one another in the absence of bound ligands, with cognate CTD added in trans forming a tight complex. The extent to which conformational switching characterizes eukaryotic COG0523s is discussed. Significance StatementThe cellular response to nutrient transition metal limitation is evolutionarily conserved in all kingdoms, providing protection from the loss of these essential inorganic cofactors that power much of metabolism. An important part of this response is the increased cell abundance of members of the enigmatic and ubiquitous Cluster of Orthologous Groups 0523 (COG0523) superfamily. In bacterial pathogens, these enzymes are often associated with the low-zinc adaptive response to host-mediated nutritional immunity, where the host deploys transition metal chelation as an innate immune response to infections. In this work, we provide new mechanistic insights into COG0523 function with the discovery of "self-gapping" by the C-terminal domain of a two-domain G-protein architecture, placed into the context of a metallochaperone model.

biochemistry↗

bioRxiv2025

Functional Characterization of Zymoseptoria tritici Candidate Effectors Reveals Their Role in Modulating Immunity in Nicotiana benthamiana

Zymoseptoria tritici is a significant wheat pathogen responsible for Septoria tritici blotch (STB) disease. Despite its economic impact, understanding of the molecular interactions between Z. tritici and its host remains limited, particularly the functions of many uncharacterized candidate effectors. Here, we assessed seven Z. tritici candidate effectors with elevated expression during the early biotrophic phase and transition to necrotrophy by transiently expressing them in the heterologous, non-host system Nicotiana benthamiana, with and without their predicted signal peptides. AlphaFold structural predictions revealed that two candidates share similarity with proteins of known function: a sterol-binding protein from Saccharomyces cerevisiae and a necrosis-inducing effector from the apple canker pathogen Valsa mali. Effector-associated phenotypes did not always correlate with expression timing, and signal peptide presence significantly influenced observed activities. Because N. benthamiana is not a natural host of Z. tritici, observed responses may reflect conserved, host-independent activities of effectors or immune recognition by N. benthamiana, rather than their native functions in wheat. Consistent with this distinction, two candidate effectors, Mycgr3107904 and Mycgr394290, induced cell death in N. benthamiana while also modulating ROS burst, likely indicative of immune recognition. Some candidates attenuate ROS production, suggesting potential targeting of conserved cellular processes, whereas none suppress PBR1-mediated cell death. Overall, our results indicate that candidate effector activities cannot be inferred from expression profiles alone, and that responses in the non-host N. benthamiana system reflect a combination of immune recognition and potential conserved activities, and primarily serve to prioritize candidates for validation in the native wheat-Z. tritici interaction.

molecular biology↗

bioRxiv2025

EZH2 alters aggressive variant prostate cancer subtype evolution, but is not required for neuroendocrine prostate cancer

Advanced prostate cancer (PrCa) remains a leading cause of cancer-related death among men because nearly all patients progress on standard therapy that targets androgen receptor (AR) signaling. An important mechanism driving therapeutic resistance is lineage plasticity, the ability of PrCa cells to reprogram into lineage variants like neuroendocrine (NE) PrCa that no longer depend on AR signaling. The histone methyltransferase EZH2 has been implicated in PrCa lineage plasticity and EZH2 inhibitors are being evaluated clinically for the treatment of PrCa. Here we test how Ezh2 impacts the evolution of PrCa lineage plasticity using genetically engineered mice and human patient data. Reduced Ezh2 expression diversifies the evolution of PrCa lineage variants, including a newly discovered variant with high expression of neurofilament genes, in both mice and humans. Lineage variant diversification is associated with activation of Klf transcription factor programs and AR cistrome reprogramming that is normally repressed by Ezh2. These findings advance the understanding of PrCa lineage plasticity and have implications for using EZH2 as a target for PrCa therapy.

cancer biology↗

bioRxiv2024

The Fusarium graminearum effector protease FgTPP1 suppresses immune responses and facilitates Fusarium Head Blight Disease

Most plant pathogens secrete effector proteins to circumvent host immune responses, thereby promoting pathogen virulence. One such pathogen is the fungus Fusarium graminearum, which causes Fusarium Head Blight (FHB) disease on wheat and barley. Transcriptomic analyses revealed that F. graminearum expresses many candidate effector proteins during early phases of the infection process, some of which are annotated as proteases. However, the contributions of these proteases to virulence remains poorly defined. Here, we characterize a F. graminearum endopeptidase, FgTPP1 (FGSG_11164), that is highly upregulated during wheat spikelet infection and is secreted from fungal cells. To elucidate the potential role of FgTPP1 in F. graminearum virulence, we generated FgTPP1 deletion mutants ({Delta}Fgtpp1) and performed FHB infection assays. While the number of completely bleached spikes infected by F. graminearum wild-type reached 50% of total infected spikes, the number of fully bleached spikes infected by{Delta} Fgtpp1 mutants was 25%, suggesting FgTPP1 contributes to fungal virulence. Transient expression of green fluorescent protein (GFP)-tagged FgTPP1 revealed that FgTPP1 localizes, in part, to chloroplasts and attenuates chitin-mediated activation of mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species production, and cell death induced by an autoactive disease resistance protein when expressed in planta. Notably, the FgTPP1 protein is conserved across the Ascomycota phylum, making it a core effector among ascomycete plant pathogens. These properties make FgTPP1 an ideal candidate for decoy substrate engineering, with the goal of engineering resistance to FHB, and likely other crop diseases caused by ascomycete fungi.

plant biology↗

bioRxiv2024

Selective promotion of oligonucleotides in the course of evolution

Evolution shapes the structure and content of genomes, yet the contribution of local sequence composition to variant selection remains poorly understood. While traditional models emphasize protein function or cross-species conservation, we propose that intra-genomic patterns of oligonucleotide (k-mer) frequencies also reflect selective forces. To explore this, we developed kGain score, a metric that quantifies the frequency shift of a k-mer upon single-nucleotide substitution, using the surrounding genomic context as a baseline. We hypothesize that variants arising in high-kGain contexts are more likely to persist due to evolutionary favorability. We validated this hypothesis across multiple systems. In E. coli and S. cerevisiae long-term evolution experiments, we found that fixed, essential, and parallel mutations consistently show elevated kGain scores. This trend held in SARS-CoV-2 variants of concern and in an in-house antibiotic adaptation experiment, where a high-kGain fusA Y515N mutation conferred resistance and maintained fitness when overexpressed, demonstrating a causal link between kGain and adaptive potential. To enable cross-species generalization, we trained a transformer-based neural network regressor on LTEE-derived mutations to predict kGain from sequence alone. The model achieved high correlation in held-out in-domain data (Pearson r = 0.81) and accurately predicted kGain trends in out-of-domain data (Pearson r = 0.82), demonstrating that k-mer-based sequence constraints learned from one genome can be effectively transferred to others. Together, our results establish kGain as a biologically meaningful, scalable metric for probing within-genome sequence selection, offering a complementary lens to existing conservation-based frameworks for understanding evolutionary fitness and variant persistence.

evolutionary biology↗

bioRxiv2024

Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogen Phyllachora maydis

Phyllachora maydis is an ascomycete foliar fungal pathogen and the causal agent of tar spot in maize. Though P. maydis is considered one of the most economically important foliar pathogens of maize, our general knowledge of the trophic lifestyle and functional role of effector proteins from this fungal pathogen remains limited. Here, we utilized a genome-informed approach to predict the trophic lifestyle of P. maydis and functionally characterized a subset of candidate effectors from this fungal pathogen. Leveraging the most recent P. maydis genome annotation and the CATAStrophy pipeline, we show this fungal pathogen encodes a predicted Carbohydrate-active enzymes (CAZymes) repertoire consistent with that of biotrophs (monomertrophs). To investigate fungal pathogenicity, we selected eighteen candidate effector proteins that were previously shown to be expressed during primary disease development. We assessed whether these putative effectors share predicted structural similarity with other characterized fungal effectors and determined whether any suppress plant immune responses. Using AlphaFold2 and Foldseek, we showed one candidate effector, PM02_g1115, adopts a predicted protein structure similar to that of an effector from Verticillium dahlia. Furthermore, transient expression of candidate effector-fluorescent protein fusions in Nicotiana benthamiana revealed that most effector proteins localize to both the nucleus and the cytosol. Importantly, three candidate effectors consistently attenuated chitin-mediated reactive oxygen species production in N. benthamiana. Collectively, these results presented herein provide valuable insights into the predicted trophic lifestyle and putative functions of effectors from P. maydis and will likely stimulate continued research to elucidate the molecular mechanisms used by P. maydis to induce tar spot.

plant biology↗

bioRxiv2023

Small-molecule degraders reduce Aβ production through CAPRIN1-mediated lysosomal degradation of APP in Alzheimer iPSC-derived neurons

Overexpression of amyloid precursor protein (APP) is a key driver of amyloid {beta} (A{beta}) pathology, making APP a compelling therapeutic target in Alzheimers disease (AD). Here, we identify small molecules that selectively degrade APP in human neurons through a targeted protein degradation approach. Structural analyses reveal that these compounds act as molecular glues, binding at the interface between cytoplasmic activation/proliferation-associate protein 1 (CAPRIN1) and APP and stabilizing their interaction within a ternary complex. In neurons derived from induced pluripotent stem cells (iPSCs) from sporadic and familial AD patients, these compounds promote CAPRIN1-dependent degradation of both wild-type and mutant APP through the endo-lysosomal pathway. The lead compound, 0152, is blood-brain barrier permeable, and its administration significantly reduces A{beta} production and amyloid burden in the brains of 5xFAD mice. These findings demonstrate that molecular glue degraders harness the CAPRIN1-dependent lysosomal pathway for selective APP degradation, offering a targeted therapeutic strategy for AD.

neuroscience↗

bioRxiv2023

Mechanisms of infection and response of the fungal wheat pathogen Zymoseptoria tritici during compatible, incompatible and non-host interactions

Zymoseptoria tritici is responsible for Septoria tritici blotch, a disease causing significant annual yield losses in wheat. To investigate infection phase-specific gene expression in the pathogen, we analyzed gene expression during infection of susceptible (Taichung 29) and resistant (Veranopolis and Israel 493) wheat cultivars, plus the non-host species barley at 1, 3, 6, 10, 17 and 23 days post inoculation (DPI). There were dramatic differences in pathogen gene expression at 10 DPI in the susceptible compared to both resistant interactions. The most pronounced differences in pathogen gene expression were observed at 3 DPI in both the susceptible and resistant host interactions compared to the non-host interaction. Thirty-one putative effectors showed early expression during the susceptible interaction compared to the non-host interaction, and six effectors were selected for subcellular localization studies. Using Agrobacterium-mediated transient expression in Nicotiana benthamiana, subcellular localization assays revealed that two candidate effectors, Mycgr3109710 and Mycgr394290, localized to putative mobile cytosolic bodies when expressed without their signal peptides, suggesting potential roles in intracellular signaling or host gene regulation. When expressed with their native signal peptides, four candidate effectors localized to the cytosol, while one effector, Mycgr3107904, did not accumulate to detectable levels, as shown by immunoblot analysis, indicating degradation. Comparison of pathogen gene expression in the susceptible host to expression in the resistant hosts, allowed us to identify genes that are expressed during the transition from biotrophic to necrotrophic growth at 10 DPI. Comparison of pathogen gene expression in resistant and susceptible hosts, versus in the non-host barley, allowed us to identify genes involved in initial colonization and host recognition. In addition, our study contributes to understanding candidate effectors that are activated early during infection and may play a role in the initial suppression of plant immunity, making them strong candidates for functional characterization.

plant biology↗

bioRxiv2023

Pseudomonas syringae pv. tomato DC3000 induces defense responses in diverse maize inbred lines

Many phytopathogens translocate virulence (effector) proteins into plant cells to circumvent host immune responses during infection. One such pathogen is Pseudomonas syringae pv. tomato DC3000, which secretes at least twenty-nine effectors into host cells, of which a subset elicits host defense responses in crop plant species. However, it is unknown whether P. syringae pv. tomato DC3000 activates immune responses in diverse maize inbreds. Here, we screened a diverse maize germplasm collection for effector-dependent recognition of this bacterial pathogen. As a control, we infiltrated Pseudomonas syringae DC3000(D36E), a derivative of P. syringae pv. tomato DC3000 that lacks all endogenous effectors. In our evaluations, we observed a variety of responses to P. syringae pv. tomato DC3000 in maize and scored the phenotypes as either no observable response (N) or as one of three responses: weak chlorosis (WC), chlorosis (C) with minimal cell death, and hypersensitive reaction (HR)-like cell death. Of the twenty-six maize inbreds screened, 13 were scored as N, 2 as WC, 2 as C, and 9 as HR-like cell death. Importantly, no maize line responded to P. syringae DC3000(D36E), demonstrating the responses observed are likely dependent upon recognition of one or more Pseudomonas effectors. Importantly, maize inbreds that recognize P. syringae pv. tomato DC3000 accumulated detectable hydrogen peroxide as well as an increase in transcript expression of a subset of maize defense genes. Collectively, our results will likely stimulate new research aimed at identifying the cognate maize disease resistance proteins that recognize the activities of one or more bacterial effectors.

plant biology↗

bioRxiv2023

A Ralstonia solanacearum type III effector alters the actin and microtubule cytoskeleton to promote bacterial virulence in plants

Cellular responses to biotic stress frequently involve signaling pathways that are conserved across eukaryotes. These pathways include the cytoskeleton, a proteinaceous network that senses external cues at the cell surface and signals to interior cellular components. During biotic stress, dynamic cytoskeletal rearrangements serve as a platform from which early immune-associated processes are organized and activated. Bacterial pathogens of plants and animals use proteins called type III effectors (T3Es) to interfere with host immune signaling, thereby promoting virulence. We previously found that RipU, a T3E from the soilborne phytobacterial pathogen Ralstonia solanacearum K60 (Rs K60), co-localizes with the plant cytoskeleton. Here, we show that RipU from Rs K60 (RipUK60) physically associates with both actin and tubulin and disrupts actin and microtubule cytoskeleton organization. We find that pharmacological disruption of the tomato (Solanum lycopersicum) cytoskeleton promotes Rs K60 colonization. RipUK60 suppresses cell surface-triggered immune responses including flg22-mediated reactive oxygen species (ROS) production and callose deposition. Importantly, tomato plants inoculated with Rs K60 lacking RipUK60 ({Delta}ripUK60) had reduced wilting symptoms and significantly reduced root colonization when compared to plants inoculated with wild-type Rs K60. Collectively, our data suggest that Rs K60 uses the type III effector RipUK60 to remodel cytoskeletal organization, thereby promoting pathogen virulence.

plant biology↗

bioRxiv2023

GRK specificity and beta gamma dependency determines a GPCR`s potential in biased agonism

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent {beta}-arrestin recruitment. Recently, it was shown that GPCRs differentially depend on GRK2/3, GRK2/3/5/6 or GRK5/6 for their regulation. The four ubiquitously expressed GRKs are classified into the cytosolic GRK2/3 and the membrane-tethered GRK5/6 subfamily. In vitro studies revealed that GRK2/3 interact with the membrane-localized G protein {beta}{gamma}-subunits. Yet, the role of this interaction as crosslink between G protein activation and {beta}-arrestin binding to GPCRs remained strongly underappreciated. Here we systematically show that the G{beta}{gamma}-GRK2/3 interaction is key for these GRKs to mediate {beta}-arrestin2 binding to Gs-, Gi- and Gq-coupled GPCRs. In our GRK2/3/5/6 knockout cells, without endogenous GRK background, the utilized GRK2/3 mutants devoid of the G{beta}{gamma} interaction site significantly diminished {beta}-arrestin2 recruitment to the beta-2 adrenergic receptor (b2AR), muscarinic M2 and M5 acetylcholine receptors (M2R, M5R). This effect was overwritten by artificially tethering GRK2/3 via a CAAX motif to the plasma membrane independently of free G{beta}{gamma}. Hence, the membrane recruitment is crucial for GRK2/3-mediated {beta}-arrestin2 binding to GPCRs, which is naturally induced via the G{beta}{gamma} interaction. This connects the {beta}-arrestin interaction for GRK2/3-regulated receptors inseparably with the associated G protein activation. We outline a theoretical framework of how GRK dependence on free G{beta}{gamma} can determine a GPCRs potential in biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we propose that it will likely be mechanistically unattainable to create {beta}-arrestin-biased ligands for the subgroup of GRK2/3-regulated GPCRs, while GRK5/6-mediated receptor regulation is independent from G{beta}{gamma} availability. Accordingly, one should first determine the GRK specificity of a GPCR to ultimately assess the receptors potential for the development of biased ligands.

pharmacology and toxicology↗

bioRxiv2023

Analysis of cell death induction by the barley NLR immune receptor PBR1

The barley (Hordeum vulgare subsp. vulgare) disease resistance protein AvrPphB Response 1 (PBR1) mediates recognition of the Pseudomonas syringae effector, AvrPphB. PBR1 belongs to the coiled-coil nucleotide-binding leucine-rich repeat (CNL) family. However, little is known about the molecular mechanisms that lead to PBR1-dependent cell death (hypersensitive reaction; HR) in response to AvrPphB. Here, we investigated PBR1 immune signaling after Agrobacterium-mediated transient expression in Nicotiana benthamiana. We found that co-expression of PBR1 with AvrPphB resulted in robust cell death, confirming previous observations that PBR1 is indeed the cognate NLR that recognizes AvrPphB. The N-terminal tagging of PBR1 with super Yellow Fluorescent Protein (sYFP) abolished PBR1-mediated cell death, demonstrating that an N-terminal epitope tag disrupts PBR1-mediated immune signaling. Furthermore, none of the individual protein domains or truncations of PBR1 induced a HR-like cell death response as strong as full-length PBR1 when co-expressed with AvrPphB, indicating that the individual domains and fragments of PBR1 are insufficient to trigger HR. Intriguingly, introducing the typically auto-activating D496V mutation within NB-ARC-containing fragments of PBR1 does not activate immune signaling revealing PBR1-mediated immune signaling requires cooperation of all domains in cis. Using co-immunoprecipitation and split-luciferase assays, we also show full-length PBR1 self-associates in the absence of AvrPphB and such self-association is not dependent on a functional P-loop/Walker A motif. Collectively, these findings provide valuable insights into PBR1-mediated disease resistance and extends upon our understanding of NLR-mediated immune signaling.

plant biology↗

bioRxiv2023

Activation Dynamics of Ubiquitin Specific Protease 7

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its "invisible" active conformation in solution, which may contribute to its activation mechanism.

biophysics↗

bioRxiv2022

Candidate effector proteins from the maize tar spot pathogen Phyllachora maydis localize to diverse plant cell compartments

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmECs) proteins remain unknown and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super Yellow Fluorescent Protein (sYFP)-tagged P. maydis effector candidate proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest effector candidate proteins from P. maydis target diverse cellular organelles and may thus provide valuable insights into their putative functions as well as host processes potentially manipulated by this fungal pathogen.

plant biology↗

bioRxiv2022

TNF signaling is required for castration-induced vascular damage preceding prostate cancer regression

The mainstay treatment for locally advanced, recurrent, or metastatic prostate cancer (PrCa) is androgen deprivation therapy (ADT). ADT causes prostate cancers to shrink in volume, or regress, by inducing epithelial tumor cell apoptosis. In normal, non-neoplastic murine prostate, androgen deprivation via castration induces prostate gland regression that is dependent on TNF signaling. Besides this direct mechanism of action, castration has also been implicated in an indirect mechanism of prostate epithelial cell death which has been described as vascular regression. The initiating event is endothelial cell apoptosis and/or increased vascular permeability. This subsequently leads to reduced blood flow and perfusion, and then hypoxia, which may enhance epithelial cell apoptosis. Castration-induced vascular regression has been observed in both normal and neoplastic prostate. We used photoacoustic, power Doppler, and contrast-enhanced ultrasound imaging, and CD31 immunohistochemical staining of the microvasculature to assess vascular integrity in the period immediately following castration, enabling us to test the role of TNF signaling in vascular regression. In two mouse models of androgen-responsive prostate cancer, TNF signaling blockade using a soluble TNFR2 ligand trap reversed the functional aspects of vascular regression as well as structural changes in the microvasculature, including reduced vessel wall thickness, cross-sectional area and vessel perimeter length. These results demonstrate that TNF signaling is required for vascular regression, most likely inducing endothelial cell apoptosis and increasing vessel permeability. Since TNF is also the critical death receptor ligand for prostate epithelial cells, we propose that TNF is a multi-purpose, comprehensive signal within the prostate cancer micro-environment mediating prostate cancer regression following androgen deprivation. SIGNIFICANCEThese studies define TNF as the mediator of androgen deprivation therapy-induced functional and structural vascular damage in prostate tumors.

cancer biology↗

bioRxiv2021

The Anaphase Promoting Complex/ cyclosome co-activator, Cdh1, is a novel target of human Papillomavirus 16 E7 oncoprotein in cervical oncogenesis

The transforming properties of the high risk human papillomavirus E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of retinoblastoma (Rb) family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase promoting complex/ cyclosome (APC/C) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the carboxyl-terminal zinc finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

biochemistry↗