Biology subjects

Kumar, P.

Publications and source records attributed to Kumar, P..

At least 181 records · Page 10Linked to original sources

bioRxiv2021

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses utilizing ACE2 as their receptor. Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbor structurally validated mutations that enhance spike (S) binding and remove angiotensin enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent-cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, it delayed death or effectively resolved lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

microbiology↗

bioRxiv2021

Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal {beta}-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.

microbiology↗

bioRxiv2021

Engineered neuron-targeting, placental mesenchymal stromal cell-derived extracellular vesicles for in utero treatment of myelomeningocele

This study investigated the feasibility and efficiency of neuron-targeting hybrid placental mesenchymal stromal cell-derived extracellular vesicles (PMSC-EVs), engineered by membrane fusion with Targeted Axonal Import (TAxI) peptide modified, TrkB agonist 7,8-DHF-loaded liposomes for treatment of myelomeningocele (MMC) via intra-amniotic cavity administration. The prepared TAxI modified liposomes with 7,8-DHF were used to fuse with PMSC-EVs. Different fusion approaches were investigated and freeze-thaw-extrude method was found to be the optimal. The engineered PMSC-EVs had a uniform particle size and efficiently loaded 7,8-DHF. It also had typical markers of native EVs. Freeze-thaw-extrude process did not change the release profile of 7,8-DHF from engineered EVs compared to TAxI modified, 7,8-DHF loaded liposomes. The engineered EVs could elicit TrkB phosphorylation depending on the incorporation of 7,8-DHF while native EVs did not. The engineered EVs increased neurite outgrowth of apoptotic cortical neurons induced by staurosporine, suggesting that they exhibited neuroprotective function. In a rodent model of MMC, neuron-targeting, engineered EVs became an active targeting delivery system to MMC defect sites. Pups treated with engineered EVs had the lowest density of apoptotic cells and displayed a therapeutic outcome. The study suggests the potential use of engineered hybrid, active neuron-targeting EVs for the in utero treatment of MMC.

bioengineering↗

bioRxiv2021

Mitoxantrone dihydrochloride, an FDA approved drug, binds with SARS-CoV-2 NSP1 C-terminal

One of the major virulence factors of SARS-CoV-2, NSP1, is a vital drug target due to its role in host immune evasion through multiple pathways. NSP1 protein is associated with inhibiting host mRNA translation by binding to the small subunit of ribosome through its C-terminal region. Previously, we have shown the structural dynamics of NSP1 C-terminal region (NSP1-CTR) in different physiological environments. So, it would be very interesting to investigate the druggable compounds that could bind with NSP1-CTR. Here, in this article, we have performed the different spectroscopic technique-based binding assays of an anticancer drug Mitoxantrone dihydrochloride (MTX) against the NSP1-CTR. We have also performed molecular docking followed by computational simulations with two different forcefields up to one microsecond. Overall, our results have suggested good binding between NSP1-CTR and MTX and may have implications in developing therapeutic strategies targeting NSP1 protein of SARS-CoV-2.

biophysics↗

bioRxiv2021

An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice

Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

microbiology↗

bioRxiv2021

Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or {beta}-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by {beta}-lactams. Here we report identification of an allosteric site at a distance of 21 [A] from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second {beta}-lactam molecule and influences binding at the catalytic site. We provide evidence that two {beta}-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-site and one covalently at the catalytic site. This dual {beta}-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new {beta}-lactam antibiotics for M. tuberculosis.

biochemistry↗

bioRxiv2021

Acropetal and basipetal cardenolide transport in Erysimum cheiranthoides (wormseed wallflower)

Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also cardenolides as defense against insect herbivory. Ten cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides (wormseed wallflower). As is predicted by the optimal defense theory, cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered cardenolide profile showed that the seed cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal cardenolide profile. Hypocotyl grafting experiments showed that the root cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of cardenolide transport into the leaves, but a mixed cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a site of cardenolide biosynthesis and therefore also the plant tissue that is most likely to be expressing the relevant biosynthetic genes.

plant biology↗

bioRxiv2021

M-CAMP™: A cloud-based web platform with a novel approach for species-level classification of 16S rRNA microbiome sequences

The M-CAMP (Microbiome Computational Analysis for Multiomic Profiling) Cloud Platform was designed to provide users with an easy-to-use web interface to access best in class microbiome analysis tools. This interface allows bench scientists to conduct bioinformatic analysis on their samples and then download publication-ready graphics and reports. The core pipeline of the platform is the 16S-seq taxonomic classification algorithm which provides species-level classification of Illumina 16s sequencing. This algorithm uses a novel approach combining alignment and kmer based taxonomic classification methodologies to produce a highly accurate and comprehensive profile. Additionally, a comprehensive proprietary database combining reference sequences from multiple sources was curated and contains 18056 unique V3-V4 sequences covering 11527 species. The M-CAMP 16S taxonomic classification algorithm was validated on 52 sequencing samples from both public and in-house standard sample mixtures with known fractions. Compared to current popular public classification algorithms, our classification algorithm provides the most accurate species-level classification of 16S rRNA sequencing data.

bioinformatics↗

bioRxiv2021

Cryo-ET of a human GBP coatomer governing cell-autonomous innate immunity to infection

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, these activities generate large supramolecular complexes that recruit immune proteins for protection. Here, we solve the native structure of a massive antimicrobial complex generated by polymerization of 30,000 human guanylate-binding proteins (GBPs) over the entire surface of virulent bacteria. Construction of this giant nanomachine takes [~]1-3 minutes, remains stable for hours, and acts as a cytokine and cell death signaling platform atop the coated bacterium. Cryo-ET of this "coatomer" revealed thousands of human GBP1 molecules undergo [~]260 [A] insertion into the bacterial outer membrane, triggering lipopolysaccharide release that activates co-assembled caspase-4. Together, our results provide a quasi-atomic view of how the GBP coatomer mobilizes cytosolic immunity to combat infection in humans. One-Sentence SummaryThousands of GBPs coat cytosolic bacteria to engineer an antimicrobial signaling platform inside human cells.

immunology↗

bioRxiv2021

A hierarchical GBP network promotes cytosolic LPS recognition and sepsis

Bacterial lipopolysaccharide (LPS) is one of the most bioactive substances known. Trace amounts trigger robust immunity to infection but also life-threatening sepsis causing millions of deaths each year. LPS contamination of the cytosol elicits a caspase-dependent inflammasome pathway promoting cytokine release and host cell death. Here, we report an immune GTPase network controls multiple steps in this pathway by genome-engineering mice to lack 7 different guanylate-binding proteins (GBPs). Gbp2-/- and Gbp3-/- mice had severe caspase-11-driven defects that protected them from septic shock. Gbp2 recruited caspase-11 for LPS recognition whereas Gbp3 assembled and trafficked the pyroptotic pore-forming protein, gasdermin D, after caspase-11 cleavage. Together, our results identify a new functional hierarchy wherein different GBPs choreograph sequential steps in the non-canonical inflammasome pathway to control Gram-negative sepsis. One-Sentence SummaryImmune GTPase network orchestrates hierarchical immunity to bacterial products in vivo

immunology↗

bioRxiv2021

Specific BRCA and immune configurations determine optimal response to platinum-based chemotherapy in triple negative breast and ovarian carcinomas

Loss of homologous recombination repair (HRR) via germline and somatic BRCA1 or BRCA2 gene mutations and via BRCA1 promoter methylation has been associated with better response to platinum agents and PARP inhibitors, in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent clinical studies is why cancers with BRCA1 promoter methylation (BRCA1meth) respond more poorly as compared to those bearing mutations in BRCA1 and BRCA2 (BRCAmut), given the biologically equivalent HRR deficiency in both states. We dissected this problem through detailed genomic analyses of primary TNBC and OvCa cohorts, as well as experimentation with patient-derived xenograft (PDX) models and genetically engineered cell lines. Using the precise genomic scar of the tandem duplicator phenotype as a precise genomic indicator of BRCA1 deficiency, we found that, in all cohorts, BRCA1mut and BRCA1meth cancers share an equivalent degree of BRCA1-linked genomic rearrangements. Nonetheless, we consistently found that patients with BRCAmut cancers, but not those with BRCA1meth cancers, had significantly better response outcomes when compared to those with BRCA proficient cancers. When fully promoter methylated BRCA1 PDX TNBCs were exposed to a single short course of platinum chemotherapy an unmethylated BRCA1 promoter allele emerged in resultant tumors associated with an increase in BRCA1 expression. A separate analysis of PDXs derived from treatment naive TNBCs featured complete methylation of the BRCA1 promoter, whereas those derived from post-chemotherapy TNBCs invariably had only partial methylation. PDXs with partial methylation were significantly associated with lower response rates to in vivo platinum-based therapy compared to those with complete promoter methylation. Using single cell clonal expansions from a partially BRCA1meth PDX, we confirmed that the reduced level of methylation was due to the demethylation of one of the BRCA1 promoter alleles and not to the outgrowth of a non-methylated clone. Clinically, analysis of primary OvCas confirmed that high levels of BRCA1 methylation were significantly associated with reduced BRCA1 gene expression whereas cancers with lower levels of BRCA1 methylation had expression levels approaching those found in BRCA1 proficient cancers. These data suggest that unlike BRCAmut cancers, where HRR deficiency is achieved via mutations that are genetically fixed, BRCA1meth cancers are highly adaptive to genotoxin exposure and more likely to recover BRCA1 expression, which may explain their poorer therapeutic response. We further found that an increased immune transcriptional signal, especially an elevated M1 macrophage signature, is associated with enhanced response to platinum-based chemotherapy only in patients with BRCA proficient cancers, in both TNBC and OvCa cohorts underscoring the importance of characterizing molecular heterogeneity to enhance predictive precision in assigning response probabilities in TNBC and OvCa.

cancer biology↗

bioRxiv2021

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among {beta}-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

microbiology↗

bioRxiv2021

Fecal Microbiota Transplantation Drives Colonic Expression of Immune Activation Genes in a Mouse Model of Antibiotic Use

Clostridioides difficile infection (CDI) is the leading hospital acquired infection in North America. While the standard treatment for CDI remains antibiotics, fecal microbiota transplantation (FMT) has gained attention as an effective therapy to prevent relapse. Previous work has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, but the effects of FMT on the gut mucosal immune response are poorly understood. Better understanding of the molecular mechanisms driven by FMT would allow for more targeted therapy against CDI. To address this important gap in knowledge, microbial community structure and host gene expression were assessed after FMT in a mouse model of antibiotic use. Administration of FMT led to a significant increase in microbial diversity and partial restoration of community structure within 48 hours of treatment. RNA sequencing of cecal tissue identified large changes in gene expression between FMT recipient and vehicle control groups. Strikingly, genes upregulated after FMT treatment were enriched in immune activation pathways, many of which were associated with pro-inflammatory immune responses. FMT also upregulated several genes associated with type 2 immunity while repressing several associated with type 3 immunity, trends that are associated with improved response to CDI. These results highlight the interplay between the intestinal microbiota and host transcriptome and identify pathways of interest for exploring the role of FMT on treatment of recurring CDI.

microbiology↗

bioRxiv2021

Short-term diet intervention alters the small non-coding RNA (sncRNA) landscape of human sperm

Offspring health outcomes are often linked with epigenetic alterations triggered by maternal nutrition and intrauterine environment. Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing the effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of paternal effects, though less is known about the existence of similar mechanisms in human sperm. Here, we first characterised the baseline sncRNA landscape of human sperm, and then studied the effects of a 6-week diet intervention on their expression profile. Baseline profiling identified 5tRFs, miRNAs and piRNAs to be the most abundant sncRNA subtypes, primarily expressed from regulatory elements like UTRs, CpG-rich regions and promoters. Expression of a subset of these sncRNAs varied with age, BMI and sperm quality of the donor. Diet intervention enriched in vitamin D and omega-3 fatty acids showed a marked increase of these nutrients in circulation and altered the sperm sncRNA expression. These included 3 tRFs, 15 miRNAs and 112 piRNAs, with gene targets involved in fatty acid metabolism, vitamin D response (LXR/RXR activation, TGF-beta and Wnt signaling), and transposable elements. These findings provide evidence that human sperms are sensitive to alterations in exposures such as diet, and sncRNAs capture the epigenetic imprint of this change. Hence changes to paternal nutrition during preconception may improve sperm quality and offspring health outcomes. To benefit future research, we developed iDad_DB, an open access database of baseline and diet-altered sncRNA in human male germline.

bioinformatics↗

bioRxiv2021

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

biophysics↗

bioRxiv2021

Investigating the conformational dynamics of SARS-CoV-2 NSP6 protein with emphasis on non-transmembrane 91-112 & 231-290 regions

The NSP6 protein of SARS-CoV-2 is a transmembrane protein, with some regions lying outside the membrane. Besides, a brief role of NSP6 in autophagosome formation, this is not studied significantly. Also, there is no structural information available till date. Based on the prediction by TMHMM server for transmembrane prediction, it is found that the N-terminal residues (1-11), middle region residues (91-112) and C-terminal residues (231-290) lies outside the membrane. Molecular Dynamics (MD) simulations showed that NSP6 consisting of helical structures, whereas membrane outside lying region (91-112) showed partial helicity, which further used as model and obtain disordered type conformation after 1.5 microsecond. Whereas, the residues 231-290 has both helical and beta sheet conformations in its structure model. A 200ns simulations resulted in the loss of beta sheet structures, while helical regions remained intact. Further, we have characterized the residue 91-112 by using reductionist approaches. The NSP6 (91-112) was found disordered like in isolation, which gain helical conformation in different biological mimic environmental conditions. These studies can be helpful to study NSP6 (91-112) interactions with host proteins, where different protein conformation might play significant role. The present study adds up more information about NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/451329v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1b6e8aborg.highwire.dtl.DTLVardef@165564eorg.highwire.dtl.DTLVardef@551f7org.highwire.dtl.DTLVardef@1485518_HPS_FORMAT_FIGEXP M_FIG C_FIG The schematic representation of NSP6 membrane topology and conformational dynamics of residue 91-112. The N-terminal and C-terminal are shown in cytoplasmic side based on the experimental evidence on coronaviruses reported by Oostra et al., 2008. The membrane anchoring domain are shown based on the TMHMM server prediction.

biophysics↗

bioRxiv2021

Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of the matricellular protein Mindin

Intratumoral heterogeneity poses a major challenge in designing effective anti-cancer strategies. Accumulating evidence suggests that this heterogeneity arises from cancer stem cells (CSCs) that also drives tumor aggressiveness and drug resistance. The stemness of CSCs are preserved by an ill-defined combination of intrinsic and external factors and is particularly intriguing since they exist within a sea of similar cells at various degrees of differentiation. In models of cutaneous squamous cell carcinoma (cSCC), we discovered a non-EMT function for the transcription factor Snail in maintaining stemness of keratinocytes. This is accomplished by the secretion of the matricellular protein Mindin from Snail expressing cells, which creates a protective niche that impedes differentiation. In an autocrine fashion, extracellular Mindin activates a Src -STAT3 pathway to reinforce the stem/progenitor phenotype and disruption of this signalling module in human cSCC attenuates tumorigenesis. The expression of Mindin in multiple carcinomas, and its critical role in cancer progression suggests that it would be a promising target for therapeutic intervention.

cell biology↗

bioRxiv2021

MRP5 and MRP9 Play a Concerted Role in Male Reproduction and Mitochondrial Function

Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency. Significance StatementMRPs are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRR5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.

genetics↗