Biology subjects

Fulzele, A.

Publications and source records attributed to Fulzele, A..

12 recordsLinked to original sources

bioRxiv2025

Envenomation leads to venom protein reduction and recovery delay in bumblebee workers

The evolution and deployment of venom systems in animals has been central to their ecological success, though not without costs. For social bees, venom plays a key role in nest defence yet for species, such as honeybees, the act of stinging generally leads to death. However, for other social bee species, such as bumblebees, where stinging is not apparently fatal, it raises the question of what, if any, are the costs associated with venom usage (envenomation). Here, we investigated proteomic changes in the venom sac, the primary reservoir for venom storage, of Bombus terrestris workers over a short time-course post-envenomation. Our analysis reveals three key findings: 1) envenomation leads to changes in the venom sac proteome with affected proteins generally reduced in the bumblebee venom sac, suggestive of a molecular cost; 2) quantified reductions of traditional venom-associated proteins post-envenomation indicate changes in venom composition post-usage; and 3) recovery of reduced venom-associated proteins post-envenomation was delayed further indicating a potential cost at the molecular level associated with usage. Overall, our findings provide new insights into the consequences of venom usage in bumblebees.

zoology↗

bioRxiv2024

Biased recruitment of H3.3 by HIRA is dictated by de-/acetylation and determines transcription memory and response

Chromatin plasticity and epigenetic memory, fundamental for eukaryotic biology, are determined by differential/regulated de novo deposition or recycling of pre-existing histones, which in turn dictate transcriptional programs. Recruitment of the histone-H3 variant, H3.3, mediated by the HIRA chaperone complex, is both causally and consequentially associated with transcription. Despite decades of work, endogenous regulatory mechanisms that differentiate between de novo deposition and recycling activities of HIRA are still unknown. Here, we have investigated the pivotal role of HIRA de-/acetylation in regulating its function. Our results unequivocally establish function separation effects of acetyl and deacetyl mimic mutations of lysine-600, vis-a-vis de novo deposition or recycling of H3.3, respectively. Importantly, we demonstrate that HIRA deacetylation-dependent biased H3.3 recycling determines transcriptional output, possibly through preferential enrichment of H3.3-K36me3. Besides unraveling tunable regulatory mechanism that governs HIRA function, we illustrate a causal link between the chaperone activity, biased recruitment of pre-existing histones, and gene expression.

molecular biology↗

bioRxiv2022

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER) where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated-degradation (ERAD) removes these clients from the ER membrane to the cytosol in a process known as retrotranslocation. Our recent work demonstrates that rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated integral membrane ERAD substrates. To survey for potential interaction partners of Dfm1, we performed protein-proximity labeling by BioID (proximity-dependent biotin identification) followed by mass spectrometry and identified several interacting proteins known to play a role in the sphingolipid biosynthesis pathway. Specifically, we found that Dfm1 physically interacts with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components and is critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. We demonstrate for the first time that Dfm1 has a role in ER export, a function that is independent of Dfm1s canonical ERAD retrotranslocation function. Specifically, we show that loss of Dfm1 results in the accumulation of phosphorylated Orm2 at the ER, suggesting a novel role for Dfm1 in controlling Orm2 export from the ER and its subsequent degradation by EGAD. Moreover, recruitment of Cdc48 by Dfm1, which is critical for its role in ERAD retrotranslocation, is dispensable for Dfm1s role in ER export. Given that the accumulation of human Orm2 homologs, ORMDLs, are associated with many maladies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

cell biology↗

bioRxiv2022

The 2',3' cyclic phosphatase Angel1 facilitates mRNA degradation during human ribosome-associated quality control

Translation elongation stalls trigger mRNA decay and degradation of the nascent polypeptide via translation-dependent quality control pathways. One such pathway, non-stop mRNA decay (NSD), targets aberrant mRNAs that lack stop codons for example due to premature polyadenylation. Here we identify Angel1, a CCR4 deadenylase homolog whose biochemical activity remains poorly defined, as a rate-limiting factor for NSD in human cells. Angel1 associates with mRNA coding regions and proteins involved in ribosome-associated quality control and mRNA decay, consistent with a factor that monitors translation elongation stalls. Depletion of Angel1 causes stabilization of reporter mRNAs that are targeted for NSD by the absence of stop codons, but not an mRNA targeted for nonsense-mediated decay. A conserved catalytic residue of Angel1 is critical for its function in NSD. Our findings identify Angel1 as a human NSD factor and suggest that Angel1 catalytic activity plays a critical role in the NSD pathway.

molecular biology↗

bioRxiv2021

The OPAQUE1/DISCORIDA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize

Formative asymmetric divisions produce cells with different fates and are critical for development. We show the myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells prior to and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 occurs during late cytokinesis, when the phragmoplast forms the nascent cell plate. Initial phragmoplast guidance in o1 is correct; however, as phragmoplast expansion continues o1 phragmoplasts become misguided. To understand how O1 contributes to phragmoplast guidance, we identified O1-interacting proteins. Maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs), which are also required for correct phragmoplast guidance, physically interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance.

plant biology↗

bioRxiv2020

Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

molecular biology↗

bioRxiv2020

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-protein Interactions

Throughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. Coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

biochemistry↗

bioRxiv2019

BTLA and PD-1 employ distinct phosphatases to differentially repress T cell signaling

T cell-mediated destruction of tumors and virus-infected cells is restricted by co-inhibitory receptors such as programmed cell death protein 1 (PD-1). Monoclonal antibodies blocking PD-1 have produced impressive clinical activity against human cancers, but durable response is limited to a minority of patients. Previous results suggest that B and T lymphocyte attenuator (BTLA), a co-inhibitory receptor structurally related to PD-1, may contribute to the resistance to PD-1 targeted therapy and co-blockade of BTLA can enhance the efficacy of anti-PD-1 immunotherapy. However, the biochemical mechanism by which BTLA represses T cell activity and to what extent the mechanism differs from that of PD-1 is unknown. Here we examine differences in the ability of BTLA and PD-1 to recruit effector molecules and regulate T cell signaling. We show that PD-1 and BTLA recruit different tyrosine phosphatases to regulate either CD28 or T cell antigen receptor (TCR)-signaling cascades. Our data reveal unexpected disparities between two structurally related immune checkpoints and two phosphatase paralogs.

cell biology↗

bioRxiv2019

NAD+-dependent deacetylase SIRT1 is essential for meiotic progression and controls repair-recombination efficiency

Meiotic components and their functions have been extensively studied. Yet, the interplay between molecular factors and regulation of their functions that is brought about by post-translational modifications, specifically (de)-acetylation, is not well characterized. SIRT1, a NAD+-dependent deacetylase has been previously shown to be necessary for spermatogenesis. However, whether it has any role to play in mammalian meiosis remains to be uncovered. Our findings identify SIRT1 as a key determinant of meiotic progression. Knocking out SIRT1 specifically in meiocytes (SIRT1{Delta}meio) led to a delay in progression through pachytene and repair of double strand breaks. Interestingly, despite these deficits, meiotic loss of SIRT1 did not affect synapsis nor did it lead to pachytene arrest or apoptosis. Moreover, our results demonstrate that SIRT1 is required for regulating crossover frequency and its absence results in higher crossover events. Therefore, our study brings to the fore a novel regulatory factor/mechanism that is necessary for coupling of synapsis and recombination. This is noteworthy since mutations in core meiotic components result in gross defects in synapsis, repair and recombination, and very few studies have reported the differential regulation of these processes. Further, exposing SIRT1{Delta}meio to low/moderate doses of (C)-irradiation indicated that SIRT1 might be involved in eliciting recombination checkpoint arrest and in its absence pachytene cells progress to diplotene stage, unlike in the SIRT1WT mice. Importantly, exogenous damage resulted in enhanced retention of (C)H2AX in SIRT1{Delta}meio diplotene cells, reiterating the critical role that SIRT1 plays in regulating repair efficiency/kinetics. Molecularly, we find that SIRT1 interacts with MRN complex and lack of SIRT1 causes hyperacetylation of several non-histone proteins including the MRN components. Given that SIRT1{Delta}meio mice mimic MRN hypomorphs, we propose that SIRT1-dependent deacetylation of these proteins is crucial for normal meiotic progression. Taken together, our study uncovers a previously unappreciated role of SIRT1 in meiotic progression.\n\nAuthor SummaryMeiosis is a key process in germ cell development that is essential for generating genetic diversity via recombination. It involves precise spatio-temporal orchestration of various molecular events such as chromosomal synapsis, repair and recombination. Whereas the core meiotic components are well known, upstream factors that might be important for regulating their functions and also couple the downstream processes are less explored. In this paper, we report that SIRT1, a NAD+-dependent deacetylase, is necessary for meiotic progression by identifying its role in coupling of synapsis and recombination. By generating a meiosis specific knockout of SIRT1, we show that its absence in spermatocytes leads to inefficient/delayed repair and progression through pachytene. We have also uncovered that SIRT1 exerts control over recombination (cross over) frequency. Interestingly, our findings demonstrate that SIRT1 provides protection against exogenous genotoxic stress possibly by eliciting meiotic checkpoints. Thus, this study provides both cellular and molecular insights into the importance of SIRT1 mediated protein deacetylation in governing meiosis in mammals.

developmental biology↗

bioRxiv2019

A newly defined cullin-RING ubiquitin ligase promotes thermotolerance as part of the Intracellular Pathogen Response in C. elegans

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode C. elegans called the Intracellular Pathogen Response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other ubiquitin ligase components. Here we use co-immunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4 and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that co-immunoprecipitate with CUL-6 and find that FBXA-158 promotes thermotolerance. In summary, we have defined the three core components and an F-box adaptor of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.\n\nSignificance StatementIntracellular pathogen infection in the nematode Caenorhabditis elegans induces a robust transcriptional response as the host copes with infection. This response program includes several ubiquitin ligase components that are predicted to function in protein quality control. In this study, we show that these infection-induced ubiquitin ligase components form a protein complex that promotes increased tolerance of acute heat stress, an indicator of improved protein homeostasis capacity. These findings show that maintaining protein homeostasis may be a critical component of a multifaceted approach allowing the host to deal with stress caused by intracellular infection.

biochemistry↗

bioRxiv2018

Deacetylation of catalytic lysine in CDK1 is essential for Cyclin-B binding and cell cycle

Cyclin-dependent-kinases (CDKs) are essential for cell cycle progression. While dependence of CDK activity on Cyclin levels is established, molecular mechanisms that regulate their binding are less studied. Here, we show that CDKl:Cyclin-B interactions are regulated by acetylation, which was hitherto unknown. We demonstrate that cell cycle dependent acetylation of the evolutionarily conserved catalytic lysine in CDK1 or eliminating its charge state abrogates Cyclin-B binding. Opposing activities of SIRT1 and P300 regulate acetylation, which marks a reserved pool of CDK1. Our high resolution structural analyses into the formation of kinase competent CDK1: Cyclin-B complex have unveiled long-range effects of catalytic lysine in configuring the CDK1 interface for Cyclin-B binding. Cells expressing acetylation mimic mutant of Cdc2 in yeast are arrested in G2 and fail to divide. Thus, by illustrating cell cycle dependent deacetylation as a determinant of CDK1:Cyclin-B interaction, our results redefine the current model of CDK1 activation and cell cycle progression.

cell biology↗

bioRxiv2018

Active protein neddylation or ubiquitylation is dispensable for stress granule dynamics

Many protein homeostasis stressors induce the formation of membraneless cytoplasmic stress granules (SGs) that contain large assemblies of repressed mRNAs and associated RNA binding proteins. Similar stressors have been shown to globally alter the function of the ubiquitin proteasome system (UPS) resulting in the accumulation of ubiquitylated proteins. Previous studies have demonstrated that ubiquitin and specific UPS components co-localize with SGs and that reducing the abundance or activity of ubiquitin pathway proteins can inhibit SG formation. These studies suggest that SG dynamics and composition may be regulated by ubiquitylation of SG resident proteins. Using ubiquitin-specific proteomic approaches, we demonstrate that many proteins, including some SG proteins are dynamically ubiquitylated upon SG-inducing sodium arsenite treatment. We utilized potent and selective inhibitors of the ubiquitin activating enzyme (UAE) or the NEDD8 activating enzyme (NAE) to directly test if active protein ubiquitylation or neddylation was required for SG dynamics. Using ubiquitin-site specific proteomics, we establish that UAE inhibition results in the rapid loss of nearly all protein ubiquitylation regardless of ubiquitin chain type. Addition of UAE or NAE inhibitors to cells did not alter arsenite-induced SG formation or dissolution. While we confirmed that ubiquitin co-localizes with both sodium arsenite and thapsigargin-induced SGs, antibodies that recognize all forms of ubiquitin more strongly co-localize with SGs compared to antibodies that preferentially recognize polyubiquitin or specific polyubiquitin-linkages. Interestingly, ubiquitin itself co-localizes with SGs in a UAE independent manner suggesting that the ubiquitin present within SGs is likely unconjugated ubiquitin. Our findings clearly demonstrate that active protein ubiquitylation or neddylation is not required for SG dynamics. These results suggest that ubiquitin-binding SG proteins may recruit free ubiquitin into SGs to modulate SG protein interactions.

cell biology↗