Biology subjects

Rai, A.

Publications and source records attributed to Rai, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

MutAIverse: An AI-Powered, Mechanism-backed Platform for Discovering Novel DNA Adducts and their precursor Genotoxins

Genotoxin exposure leads to DNA adduct formation, potentially causing mutations if unrepaired. Current DNA adductomics platforms or analytical workflows are limited by incomplete spectral libraries, reliance on experimentally validated adducts, limited cellular contexts, and inefficient computational methodologies. We introduce MutAIverse, an advanced AI-driven DNA adductomics analysis platform that overcomes these limitations by leveraging intracellular mechanistic modeling of genotoxin bioactivation to construct a comprehensive DNA adduct library and offers advanced spectral mapping. MutAIverse integrates experimentally validated and chemically valid putative DNA adducts, enabling interpretable retrograde tracking to parental genotoxins. Validation of MutAIverse against experimental MS/MS datasets demonstrated the detection of both known and novel adducts. Furthermore, application to in-house generated DNA adductomics data from tissue biopsies of smokeless tobacco-induced head and neck cancer patients revealed selective enrichment of both novel and known DNA adducts; a subset of them was validated using MS/MS analysis. Collectively, MutAIverse provides a robust, end-to-end, and interpretable platform for advanced DNA adductomics analysis.

bioinformatics↗

bioRxiv2025

Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Midbody Remnant Proteins Following Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process that allows highly polarized, immotile epithelial cells to transform into motile mesenchymal cells: it is a fundamental cellular process involved in embryonic development, tumour cell metastasis, organ fibrosis and tissue regeneration. To assess the contribution of secreted midbody remnants (MBRs) - a new class of membranous extracellular vesicle (EV) molecularly distinct from exosomes/small EVs - to the EMT process, we conducted a proteomic analysis of MBRs released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). MBRs were harvested from cell culture media in milligram quantities using a continuous culture bioreactor device and purified using sequential centrifugation and buoyant density gradient centrifugation (OptiPrep). Gel-MS/MS protein profiling showed MDCK cell-MBRs reflect their epithelial origin (e.g., enriched CDH1, DSP, THBS1, OLCN, EPCAM proteins) and 21D1 cell-MBRs their mesenchymal phenotype (e.g., HRAS, VIM, MMP14, CDH2, WNT5A and enriched invasive and cell motility proteins). Prominent findings were the unique expression of the immune checkpoint protein NT5E/CD73 (ecto-5'-nucleotidase), and ser/thr kinases LIMK1/K2 in 21D1-MBRs (not present in MDCK cell-MBRs), and enrichment in Wnt signalling network proteins. Collectively, our findings suggest MBRs might play a previously unrecognized role in the EMT process. SignificanceO_LIEpithelial-to-mesenchymal transition (EMT) is a critical cell biological process that occurs during normal embryonic development and cancer progression. Our study describes, for the first time, the large-scale sequential purification of secreted midbody remnants (MBRs) and exosomes/sEVs from the in vitro cell line EMT model Madin-Darby canine kidney (MDCK) cells and MDCK cells transformed with oncogenic H-Ras (21D1 cells): GeLC-MS/MS protein profiling identified the repertoire of enriched MDCK-MBR proteins following EMT. C_LIO_LIMBRs display a proteome profile distinct from sEVs that is enriched with factors of the centralspindlin complex (KIF23.1, KIF4A, INCENP, CEP55, PLK1) and further include components of the mitochondrial network, cytokinesis, microtubule movement, and intercellular connection. C_LIO_LIIn the context of EMT, our data reveal simultaneous activation of EMT signalling pathways in MBRs including signalling receptor binding, regulation of cell differentiation, and Wnt, VEGF and PDGF signalling. C_LIO_LIWe identify several mesenchymal enriched networks in MBRs associated with focal adhesion, cell matrix, kinase activity, and cell shape/organisation, while epithelial derived MBRs are show enriched networks predominately associated with mitochondrial (processing/transport), midbody, and plasma membrane annotation. C_LIO_LIOur study sheds light on the signalling architecture of MBRs following oncogenic H- Ras-induced EMT: collectively, our data informs ongoing efforts to delineate oncogenic drivers of cancer initiation, progression, and metastasis. C_LI

cell biology↗

bioRxiv2025

Consistency in floral volatiles impacts plant-pollinator interactions in the face of environmental change in the Eastern Himalayas

Floral scents are an important mediator of many plant-pollinator interactions. However, environmental and ecological change could affect the synthesis and emission of volatile chemicals that constitute floral scents, potentially influencing pollinator perception of scents. We investigated the effects of elevation and warming on the floral scents of multiple co-occurring alpine meadow plant species along an elevational gradient in the Eastern Himalayas (3000 - 4000 masl). We also investigated pollinator responses in the same locations using 3-D printed floral models that replicated the differing volatile constituents. Finally, we assessed how pollinator preferences changed with variations in elevation, flowering season and location. We found that elevation and in-situ warming using Open Top Chambers (OTC) led to significant intraspecific variation in the composition of floral scents, both in terms of quality and quantity of volatiles released. Nevertheless, conspecific floral scents across elevations were more similar to each other than to other species. Cafeteria assays using artificial floral models revealed that pollinator preferences were driven by a small number of volatiles (p-cymene, 2-pentylfuran and -pinene), but these preferences were abolished in a novel plant-pollinator community where the same floral volatiles were not detected. Our results suggest that the local odourscape established by the floral community present plays a key role in plant-pollinator interactions. This emphasizes the need to incorporate ecological impacts such as changes in community into climate change analyses to understand the long-term impacts of human activity on co-evolutionary relationships like pollination. Significance StatementFloral scents are important for most plant-pollinator interactions, and can be affected by environmental factors like elevation and temperature. Our study shows that elevation and simulated in situ warming change the composition of floral scents, yet scents from the same species were more similar to each other across elevations and temperatures than to other species. Further, pollinator preferences to these scents replicated in artificial models persisted in spite of variation in temperature and elevation, but not in a novel plant-pollinator community containing floral species not releasing those volatiles. This emphasizes the need to incorporate factors such as floral community composition into our understanding of the long-term impacts of climate change and human activity on co-evolutionary relationships like pollination.

ecology↗

bioRxiv2025

HP-Bodies - Ancestral Condensates that Regulate RNA Turnover and Protein Translation in Bacteria

Uncovering what drives select biomolecules to form phase-separated condensates in vivo and identifying their physiological significance are topics of fundamental importance. Here we show that nitrogen-starved Escherichia coli produce long-chain polyphosphates, which scaffold the RNA chaperone Hfq into phase-separating high molecular weight complexes together with components of the RNA translation and processing machinery. The presence of polyphosphate within these condensates, which we termed HP-bodies, controls Hfq function by selectively stabilizing polyadenylated RNAs involved in transcription and protein translation, and promoting interactions with translation- and RNA-metabolism-associated proteins involved in de novo protein synthesis. Lack of polyphosphate prevents HP-body formation, which increases cell death and significantly hinders recovery from N-starvation. In functional analogy, we demonstrate that polyP contributes specifically to the formation of Processing (P)-bodies in human cell lines, revealing that a single, highly conserved and ancestral polyanion serves as the universal scaffold for functional phase-separated condensate formation across the tree of life.

microbiology↗

bioRxiv2024

CRISPR-Cas9 mediated deletions of FvMYB46 reduces fruit set and biosynthesis of flavonoids in Fragaria vesca

Secondary metabolites produced by the phenylpropanoid pathway, which is regulated by transcription factors of the MYB family, play crucial roles in this early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure and lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca specific U6-promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in frame 81-bp deletion of the first conserved MYB-domain or an out of frame 82-bp deletion potentially knocking out the gene function. In both types of mutant plants, pollen germination and the frequency of flowers developing to mature berries was significantly reduced compared to wild type. Transcriptomic analysis of flowers demonstrated that FvMYB46 is positively regulating the expression of genes involved in pollen germination, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis, while has a negative impact on carbohydrate metabolism. In FvMYB46-mutant flowers, the flavonols and flavan-3-olscontent, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside were reduced, and we observed a local reduction of lignin content in anthers. Together these results suggest that MYB46 control fertility and efficient fruit set by regulating cell wall structure, flavonoid biosynthesis, carbohydrate metabolism and ROS-signaling in flowers and early fruit development in F. vesca.

plant biology↗

bioRxiv2024

Biochemical and structural insights into the auto-inhibited state of Mical1 and its activation by Rab8

Mical1 regulates F-actin dynamics through the reversible oxidation of actin, a process controlled by its interactions with various proteins. Upon binding to Rab8 family members, Mical1 links endosomes to the cytoskeleton, promoting F-actin disassembly. In the absence of Rab, Mical1 exists in an auto-inhibited state, but its biochemical characterization remains incomplete. Our study reveals that the N-terminal MO-CH-LIM domains of Mical1 form an intramolecular complex with its C-terminal bMERB domain. Mutational analysis, guided by the AlphaFold2 model, identifies critical residues at the binding interface. Additionally, we demonstrate that full-length Mical1 binds to Rab8 in a 1:2 stoichiometry, thereby releasing auto-inhibition. Through structure-based mutational studies, we uncover allostery between the N and C-terminal Rab binding sites. Notably, Rab binding at the high-affinity C-terminal site precedes binding at the N-terminal site, suggesting a sequential binding mode. These findings elucidate how Rab8 binding releases the MO-CH-LIM domains from the Mical1 bMERB domain, facilitating interactions with other proteins and the actin cytoskeleton, thereby modulating actin dynamics.

biochemistry↗

bioRxiv2024

Plant species richness and the root economics space drive soil fungal communities

Trait-based approaches have been increasingly used to relate plants to soil microbial communities. However, the plant organs mediating this plant-microbe interaction - the roots - have been largely overlooked. The recent discovery of the root economics space offers a predictive framework for the structure of soil microbial communities, and specifically soil-borne fungal communities. Applying this novel approach, our study in a grassland plant diversity experiment reveals distinct root trait strategies at the level of the plant community. In addition to significant effects of plant species richness, we show that both axes of the root economics space - the collaboration and conservation gradient - are strong drivers of the composition of the different guilds of soil fungi, including saprotrophic, plant pathogenic, and mycorrhizal fungi. Our results illustrate that the root economics space and plant species richness jointly determine the effects of plants on fungal communities and their potential role in plant health and ecosystem functioning.

ecology↗

bioRxiv2024

Multi-omics discovery of hallmark protein and lipid features of circulating small extracellular vesicles in humans

Extracellular vesicles (EVs) are now being increasingly recognized as an essential signaling entity in human plasma, linking them to health and various diseases. Still, their core protein and lipid componentry, which lie at the center of EV form and function, remain poorly defined. Achieving this unmet milestone remains greatly hindered by abundant non-vesicular extracellular plasma components (non-EVs) in mass spectrometry-based analyses. Here, we performed high-resolution density gradient fractionation of over 140 human plasma samples to isolate circulating EVs, and systematically construct their quantitative proteome (4500 proteins) and lipidome (829 lipids) landscapes. This led to the discovery of a highly conserved panel of 182 proteins (ADAM10, STEAP23, STX7) and 52 lipids (PS, PIPs, Hex2Cer, PAs), providing a deep survey of hallmark molecular features and biological pathways intrinsic to circulating EVs. We also mapped the surfaceome diversity, identifying 151 proteins on the EV surface. We further establish a set of 42 proteins and 114 lipids features that served as hallmark features of non-EV particles in plasma. We submit ADAM10 and PS(36:1) as conserved EV biological markers that precisely differentiate between EV and non-EV particles. Our findings, which can be explored via an open-source Shiny web tool (evmap.shinyapps.io/evmap/) will serve as a valuable repository to the research community for a clearer understanding of circulating EV biology.

cell biology↗

bioRxiv2023

A Tumour-Specific Molecular Network Promotes Tumour Growth in Drosophila by Enforcing a JNK-YKI Feedforward Loop

Cancer cells expand rapidly in response to altered intercellular and signalling interactions to achieve hallmarks of cancer. Impaired cell polarity combined with activated oncogenes is known to promote several hallmarks of cancer e.g., activating invasion by increased activity of Jun N-terminal kinase (JNK), and sustained proliferative signalling by increased activity of Hippo effector Yorkie (Yki). Thus, JNK, Yki, and their downstream transcription factors have emerged as synergistic drivers of tumour growth through pro-tumour signalling and intercellular interactions like cell-competition. However, little is known about the signals that converge onto JNK and Yki in tumour cells that enable the tumour cells to achieve hallmarks of cancer. Here, using mosaic models of cooperative oncogenesis (RasV12, scrib-) in Drosophila, we show that RasV12, scrib- tumour cells grow by activation of a previously unidentified network comprising Wingless (Wg), Dronc, JNK and Yki. We show that RasV12, scrib- cells show increased Wg, Dronc, JNK, and Yki signalling, and all of these signals are required for the growth of RasV12, scrib- tumours. We report that Wg and Dronc converge onto a JNK-Yki self-reinforcing positive feedback signal-amplification loop that promotes tumour growth. We found that Wg-Dronc-Yki-JNK molecular network is specifically activated in polarity-impaired tumour cells and not in normal cells where apical basal polarity is intact. Our findings suggest that identification of molecular networks may provide significant insights about the key biologically meaningful changes in signalling pathways, and paradoxical signals that promote Tumourigenesis.

cancer biology↗

bioRxiv2023

Oral administration of a specific p300/CBP Lysine acetyltransferase activator induces synaptic plasticity and repairing spinal cord injury

TTK21 is a small molecule activator of p300/CBP acetyltransferase activity, which upon conjugation with glucose-derived carbon nanosphere (CSP) can efficiently cross the blood-brain barrier and activate histone acetylation in the brain. Its role in adult neurogenesis and retention of long-term spatial memory upon intraperitoneal (IP) administration is well established. In this study, we have successfully demonstrated that CSP-TTK21 can be effectively administered via oral gavage. Using a combination of molecular biology, microscopy, and electrophysiological techniques, we systematically investigated the comparative efficacy of oral administration of CSP and CSP-TTK21 in wild-type mice, evaluating their functional effects in comparison to intraperitoneal (IP) administration. Our findings indicate that CSP-TTK21, when administered orally, induces long-term potentiation in the hippocampus without significantly altering basal synaptic transmission, a response comparable to that achieved through IP injection. Remarkably, in a spinal cord injury model, oral administration of CSP-TTK21 exhibits equivalent efficacy to IP administration. Furthermore, our research demonstrates that oral delivery of CSP-TTK21 leads to improvements in motor functions, histone acetylation dynamics, and increased expression of regeneration associated genes (RAGs) in a spinal injury rat model, mirroring the effectiveness of IP administration. Collectively, these results underscore the potential utility of CSP as an oral drug delivery system, particularly for targeting the neural system.

neuroscience↗

bioRxiv2023

Rho of plant GTPases with geranylgeranylation motif modulate monoterpene indole alkaloid biosynthesis in Catharanthus roseus

Rho Of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Catharanthus roseus, indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C- terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, both of their transcripts showed higher expression in most parts compared to other CrROPs. Protein- protein interaction studies revealed that both CrROP3 and CrROP5, but not CrROP2 (lacking CSIL motif), interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of {Delta}CrROP3 and {Delta}CrROP5 mutants lacking the CSIL motif failed to enhance MIA biosynthesis. Taken together, these results implicate that CrROP3 and CrROP5 have positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

plant biology↗

bioRxiv2023

Distinct composition of plasma virome in HIV-infected subjects on antiretroviral therapy compared to controls

Microbiome study during HIV infection is widely documented with major emphasis on bacteriome, while virome studies are few. The alteration of plasma virome is reported in HIV patients who are either treatment naive or have a weak immune system. Less is known about the plasma virome in HIV-infected patients on ART with preserved CD4 counts. In our pilot study, viral DNA isolated from plasma was sequenced on Illumina Nextseq500 platform. With the help of VIROMATCH pipeline, we observed that the plasma virome of HIV patients were significantly distinct from controls on the basis of Bray-Curtis dissimilarity index. The species, Human gammaherpesvirus 4 and families, Herpesviridae and Siphoviridae were found to be significantly enriched and differentially abundant in HIV patients. Hence, plasma virome is an important component for future study that might influence disease progression among HIV patients during therapy.

microbiology↗

bioRxiv2023

TREM2 and APOE do not modulate phagocytic clearance of dying cells in the live mammalian brain

TREM2 and APOE are two major risk factors for Alzheimers disease (AD) that have been proposed to play crucial roles in microglia pathophysiology by affecting their ability to phagocytose cellular debris or aggregated proteins. In this study, we investigated for the first time the impact of TREM2 and APOE on the removal of dying neurons in the live brain by implementing a targeted photochemical method for programmed cell death induction combined with high-resolution two-photon imaging. Our findings showed that the deletion of either TREM2 or APOE did not affect the dynamics of microglia engagement with dying neurons or their efficiency in phagocytosing corpses. Interestingly, while microglia that encapsulate amyloid deposits were capable of phagocytosing dying cells without disengaging from plaques or moving their cell bodies; in the absence of TREM2, microglia cell bodies were observed to readily migrate towards dying cells, further disengaging from plaques. Our data suggest that TREM2 and APOE variants are unlikely to increase risk of AD through impaired corpse phagocytosis. SummaryHigh-resolution two-photon imaging of programmed cell death in the live mouse brain reveals that neither TREM2 nor APOE modulate microglia phagocytosis of neuronal corpses. However, TREM2 regulates microglia migratory behavior towards dying cells in the vicinity of amyloid plaques.

neuroscience↗

bioRxiv2023

Discovery of 1,3,4-oxadiazoles with slow-action activity against Plasmodium falciparum malaria parasites

To achieve malaria eradication, new preventative agents that act differently to front-line treatment drugs are needed. To identify potential chemoprevention starting points we screened a sub-set of the CSIRO Australia Compound Collection for compounds with slow-action in vitro activity against Plasmodium falciparum. This work identified N,N-dialkyl-5-alkylsulfonyl-1,3,4-oxadiazol-2-amines as a new antiplas-modial chemotype (e.g., 1 96 h IC50 550 nM) with a different action to delayed-death slow-action drugs. Structure activity relationship analysis of analogues identified multiple compounds with potent and selective in vitro activity against drug-sensitive and multi-drug resistant Plasmodium parasites (e.g., 31 and 32 96 h IC50 <40 nM; SI >2,500). However subsequent studies in mice with lead compound 1, which had the best microsomal stability of the compounds assessed, demonstrated rapid clearance (T1/2 <1.6 h) and poor oral in vivo efficacy. This indicates that improvements in the pharmacokinetic profile of N,N-dialkyl-5-alkylsulfonyl-1,3,4-oxadiazol-2-amines would be needed for the development of this chemotype for malaria chemoprophylaxis.

microbiology↗

bioRxiv2022

Neuroprotective potential of an Ayurvedic compound "Ras Sindoor" on Drosophila melanogaster model of Parkinson's disease

Parkinsons Disease (PD) is the second most common neurodegenerative disease affecting 1-2% of the global population with no cure to date. PD is characterized by accumulation of Lewy Bodies (LBs), which are caused due to aggregation of incorrectly folded -Synuclein, (SNCA). Another form of PD manifestation is characterized by loss of function of parkin, which encodes an E3 ubiquitin ligase. Despite extensive research, the cause for onset and progression of PD remains unknown and current therapeutics mainly help manage the disease. An alternative line of treatment can be useful. In this study, we have employed two different genetic models of Drosophila to screen for Ayurvedic compounds and found an Ayurvedic mercury based organo-metallic drug Ras-Sindoor has neuroprotective function. Our data indicate that characteristic locomotory dysfunction phenotype of PD is restored upon administration of the compound. Interestingly, RS fed flies also exhibit reduced transcript levels of initiator caspase dronc, which possibly prevents cell death in dopaminergic neurons. Additionally, RS fed PD model flies exhibit an enhanced life span. Our studies emphasize beneficial use of traditional Ayurvedic compounds as a holistic cure for PD like multifactorial diseases.

neuroscience↗

bioRxiv2022

Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors

Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.

cancer biology↗

bioRxiv2022

Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay

Cardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by slowing shortening velocity and sensitizing the thin filament to calcium. cMyBP-C has been shown to interact with the proximal myosin S2 tail and the thin filament. However, the relative contribution of these interactions to the collective modulation of actomyosin ensemble function remains unclear. Hence, we developed a "nanosurf" assay as a model system to interrogate cMyBP-C interactions with actin and/or myosin. Synthetic thick filaments were generated using recombinant human {beta}-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14 or 28 nm spacing, corresponding to the 14.3 nm myosin spacing found in native thick filaments. In vitro motility assays with myosin bound to the surrounding surface, exhibit enhanced thin filament interactions with synthetic thick filaments. No significant differences were observed in mean thin filament velocities between 14 and 28 nm spacing, consistent with our previous results for myosin V, VI, and {beta}-cardiac myosin S1. Our nanosurf assay demonstrates the slowing of actomyosin motility by cMyBP-C. Alternating {beta}-cardiac myosin HMM and cMyBP-C N-terminal fragments, C0-C2 or C1-C2, every 14 nm on the nanotube, reduced the mean thin filament velocity 4-6 fold relative to myosin alone. Interestingly, similar inhibition was observed using a {beta}-cardiac myosin S1 construct, which lacks the S2 region proposed to interact with cMyBP-C, suggesting the actin-cMyBP-C interactions may dominate the inhibitory mechanism. No significant inhibition of thin filament velocity was observed with a C0-C1f fragment, lacking the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A phosphomimetic C0-C2 fragment showed a 3-fold higher velocity compared to its phosphonull counterpart, further highlighting phosphorylation-dependent regulation via the M-domain. Together, we have established the nanosurf assay as a tool to precisely manipulate spatially dependent cMyBP-C binding partner interactions, shedding light on the molecular regulation of {beta}-cardiac myosin contractility. STATEMENT OF SIGNIFICANCECardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated protein associated with hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death. Despite the importance of cMyBP-C in cardiac contractility, the mechanisms underlying this regulation are unclear due to experimental challenges in studying the complex, transient, weak interactions of cMyBP-C with the contractile proteins of the sarcomere. In this study, we created a nanosurf synthetic DNA thick filament assay to dissect the cMyBP-C interactions with actin and human {beta}-cardiac myosin. We demonstrate actomyosin inhibition by cMyBP-C fragments regardless of recombinant human {beta}-cardiac myosin subfragment (HMM or S1) and highlight the importance of the cMyBP-C M-domain using cMyBP-C fragments and phosphomimetics.

biophysics↗

bioRxiv2021

miRNA mediated downregulation of non-muscle Cyclase associated protein 1 is required for myogenic differentiation

Myoblast fusion is crucial for the formation, growth and regeneration of healthy skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Here we report that Cyclase-associated protein 1 (Cap1), a regulator of actin dynamics, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in murine C2C12 and human LHCN-M2 myoblasts, but its abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of Cap1 by overexpression or Crispr-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes and myosin heavy chain expression. Endogenous Cap1 expression is posttranscriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133 and miR-206, which have conserved binding sites in the 3 UTR of the Cap1 mRNA. Deletion of the endogenous 3 UTR in C2C12 cells phenocopies overexpression of Cap1 by inhibiting myotube formation. Our findings implicate Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

cell biology↗