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Ramachandran, R.

Publications and source records attributed to Ramachandran, R..

At least 37 records · Page 2Linked to original sources
bioRxiv2025

Regulation of the lncRNA malat1/Egr1 Axis by Wnt, Notch and TGF-β signaling: A Key Mechanism in Retina Regeneration

Adult zebrafish retinas rely on the resident Muller glia to maintain homeostasis and enable regeneration. Retina regeneration remains incomplete in mammals despite extensive efforts to emulate zebrafish regenerative conditions. Many studies have examined the reprogramming of zebrafish Muller glia cells, which is necessary for regeneration driven by regeneration-associated gene expression. Here, we show that the lncRNA malat1, crucial for many biological functions, plays essential roles during retina regeneration. We demonstrate that malat1 functions through an Egr-dependent axis, modulated by Wnt, Notch, and TGF-{beta} signaling pathways, and is necessary for effective retina regeneration. Moreover, we uncover that the antisense lncRNA talam1, which regulates malat1 availability, is differentially regulated in zebrafish and mice, highlighting species-specific gene regulatory mechanisms after retinal injury. Cells with active TGF-{beta} signaling stabilize Malat1 in mice while the same signaling destabilizes malat1 in zebrafish. Taken together, our work uncovers a new role for the malat1/Egr1 axis in necessitating retina regeneration, which may have important implications for differential regenerative ability in vertebrates.

cell biology↗
bioRxiv2024

AlphaFold prediction and analysis of Adhesion-family G protein coupled receptor (aGPCR) structures.

Adhesion GPCRs (aGPCRs), a family of 33 receptors, regulate crucial physiological processes through a unique self-activation mechanism. We employed AlphaFold, a state-of-the-art AI system, to predict 3D structures of aGPCRs in active and inactive conformations. Full-length models were obtained from the AlphaFold Protein Structure Database, while tethered-ligand (TL) exposed structures were predicted using AlphaFold. Comparison with available experimentally solved structures were made to validate accuracy of Alphafold aGPCR model. For receptors without experimentally solved structures, we compare structures of inactive (TL masked) and active (TL exposed) aGPCRs. In the active structures, the tethered-ligand was docked in the transmembrane bundle for several receptors but surprisingly not all receptors. This AI driven analysis of aGPCR structures provides a framework for exploring alternative receptor activation mechanisms. These models also offer valuable insights into receptor conformational changes and TL interactions, paving the way for the development of novel pharmacological tools and accelerating drug discovery efforts targeting this important receptor family.

bioinformatics↗
bioRxiv2024

Recognizing amino acid sidechains in a medium resolution cryo-electron density map

Building an accurate atomic structure model of a protein into a cryo-electron microscopy (cryo-EM) map at worse than 3 [A] resolution is difficult. To facilitate this task, we devised a method for assigning the amino acid residue sequence to the backbone fragments traced in an input cryo-EM map (EMSequenceFinder). EMSequenceFinder relies on a Bayesian scoring function for ranking 20 standard amino acid residue types at a given backbone position, based on the fit to a density map, map resolution, and secondary structure propensity. The fit to a density is quantified by a convolutional neural network that was trained on [~]5.56 million amino acid residue densities extracted from cryo-EM maps at 3-10 [A] resolution and corresponding atomic structure models deposited in the Electron Microscopy Data Bank (EMDB). We benchmarked EMSequenceFinder by predicting the sequences of 58,044 distinct L-helix and {beta}-strand fragments, given the fragment backbone coordinates fitted in their density maps. EMSequenceFinder identifies the correct sequence as the best-scoring sequence in 77.8% of these cases. We also assessed EMSequenceFinder on separate datasets of cryo-EM maps at resolutions from 4 to 6 L. The accuracy of EMSequenceFinder (63.5%) was better than that of three tested state-of-the-art methods, including findMysequence (45%), ModelAngelo (27%), and sequence_from_map in Phenix (12.9%). We further illustrate EMSequenceFinder by threading the SARS-CoV-2 NSP2 sequence into eight cryo-EM maps at resolutions from 3.7 to 7.0 [A]. EMSequenceFinder is implemented in our open-source Integrative Modeling Platform (IMP) program. Thus, it is expected to be helpful for integrative structure modeling based on a cryo-EM map and other information, such as models of protein complex components and chemical crosslinks between them. EMSequenceFinder is available as part of our open source IMP distribution at https://integrativemodeling.org/.

biophysics↗
bioRxiv2024

TECPR2 is a Rab5 effector that regulates endosomal cargo recycling

Small GTP-binding (G) proteins of the Rabs, Arfs, and Arls (Arf-like) family mediate the recruitment of their effectors to subcellular membrane-bound compartments, which in turn mediates vesicle budding, motility, and tethering. Here, we report that Tectonin-{beta}-propeller repeat containing protein 2 (TECPR2), a protein mutated in a form of hereditary sensory and autonomic neuropathy (HSAN), is an effector of early endosomal Rab protein, Rab5, and interacts with Rab5 via its C-terminal TECPR repeats. The HSAN-associated TECPR2 (R1336W) missense variant was deficient in Rab5-binding and, consequently, in membrane recruitment. TECPR2-depletion led to perinuclear collapse of recycling endosomes and increased overlap of sorting and degradative subdomain markers on early endosomes. Consistent with a possible role in endocytic recycling, we observed impaired recycling and increased lysosomal degradation of 5{beta}1 integrin receptors upon TECPR2 knockdown. TECPR2 regulates the early endosomal localization of the cargo adaptor for {beta}1 integrins, SNX17, and its-associated protein complex WASH, which mediates actin polymerization on early endosomes. Finally, TECPR2 depletion in the zebrafish model resulted in delayed motility and changes in the neuromuscular junction. Our study supports an early endosomal role for TECPR2 in cargo recycling and provides insights into how its loss-of- function results in a neurodegenerative genetic disorder.

cell biology↗
bioRxiv2023

Structural Basis for Regulated Assembly of the Mitochondrial Fission GTPase Drp1

Mitochondrial fission is crucial for distributing cellular energy throughout cells and for isolating damaged regions of the organelle that are targeted for degradation. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. De novo missense mutations in the Drp1 gene, DNM1L, are associated with severe neurodevelopmental diseases in patients, and no effective treatments are available. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the regulatory mechanisms that determine when and where Drp1 self-assembles, and proper mitochondrial dynamics requires correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length, native Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intermolecular interactions to achieve the assembly competent state previously observed in crystal and filament structures. Specifically, the G domain is closed against the stalk domain and occludes intermolecular interactions necessary for self-assembly beyond a dimer. Similarly, adjacent stalks in the dimer form a more continuous interface that further occludes conserved intermolecular contact sites. This structural insight provides a novel mechanism for regulated self-assembly of the mitochondrial fission machinery.

cell biology↗
bioRxiv2022

dnaE2 expression promotes genetic diversity and bacterial persistence in mycobacterial biofilms

The complex cellular architecture and the microenvironments within the biofilm give rise to a population that is both physiologically and genetically heterogeneous. Transcriptome analysis of Mycobacterium smegmatis in biofilm culture and its transition phase into planktonic growth was performed to identify the genetic basis of heterogeneity in the biofilm. While there was an increase in the expression of mycobacterial mutasome consisting of dnaE2, imuA, and imuB in the biofilm, the mutation burden was less compared to the planktonic culture. Deletion of dnaE2 causes lower mutation frequency and bacterial fitness in comparison to the parental strain in biofilm culture. The expression of dnaE2 contributes to a slower bacterial growth rate, potentially promoting persister formation. Our study uncovers the multiple benefits of dnaE2 expression in biofilm such as increasing genetic diversity and reducing growth rate; both of which are necessary for mycobacterial survival and adaptation.

microbiology↗
bioRxiv2022

Autocrine Proteinase Activated Receptor (PAR) mediated signaling in prostate cancer cells.

Proteinase activated receptors (PARs) are G protein-coupled receptors (GPCRs) activated by limited N-terminal proteolysis. A variety of proteolytic enzymes derived from the coagulation cascade and inflammatory milieu activate PARs, however specific activators in different physiological and pathophysiological contexts remain poorly defined. PARs are highly expressed in many cancer cells and regulate various aspects of tumor growth and metastasis. Endogenous proteinases that regulate PARs in the setting of various tumors however remains unresolved. Prostate cancer (PCa) remains a major cause of mortality in men despite advances in early detection and clinical intervention. PAR expression has been reported in PCa, however, their role here remains poorly defined. In androgen independent PC3 cells, we find functional expression of PAR1 and PAR2 but not PAR4. Using genetically encoded PAR cleavage biosensors, we find that PCa cells secrete proteolytic enzymes that cleave PARs and trigger autocrine signaling. Deletion of PAR1 and PAR2 using CRISPR/Cas9 combined with microarray analysis revealed genes that are differentially regulated by this autocrine signalling mechanism. Interestingly, several genes that are known PCa prognostic factors or biomarker were differentially expressed in PAR1-KO and PAR2-KO PC3 cells. We also examined PAR1 and PAR2 regulation of PCa cell proliferation and migration using PAR1 and PAR2-KO PC3 cells, as well as PAR1 and PAR2 specific agonists and antagonists. We find that PAR1 and PAR2 have opposite effects on PC3 cell proliferation and migration. In summary, we have identified an autocrine signaling mechanism through PARs as a regulator of PCa cell function.

cancer biology↗
bioRxiv2022

Psychometric and subcortical neurometric measures of temporal discrimination in rhesus macaques

Temporal envelope fluctuations are abundant in nature and are critical for perception of complex sounds. While psychophysical sinusoidal amplitude modulation (SAM) processing studies have characterized the perception of SAM, and neurophysiological studies report a subcortical transformation from temporal to rate-based code, no studies have characterized this transformation in unanesthetized animals or in nonhuman primates. To address this, we recorded single-unit responses and compared derived neurometric measures in the cochlear nucleus (CN) and inferior colliculus (IC) to psychometric measures of modulation frequency (MF) discrimination in macaques. IC and CN neurons often exhibited tuned responses to SAM in their rate and spike-timing. Neurometric thresholds spanned a large range (2-200 Hz {Delta} MF). The lowest 40% of IC thresholds were less than or equal to psychometric thresholds, regardless of which code was used, while CN thresholds were greater than psychometric thresholds. Discrimination at 10-20 Hz could be explained by indiscriminately pooling 30 units in either structure, while discrimination at higher MFs was best explained by more selective pooling. This suggests that pooled brainstem activity was sufficient for AM discrimination. Psychometric and neurometric thresholds decreased as a function of stimulus duration, but IC and CN thresholds were greater and more variable than behavior at durations less than 500 ms. This slower subcortical temporal integration compared to behavior was consistent with a drift diffusion model which reproduced individual differences in performance and can constrain future neurophysiological studies of temporal integration. These measures provide an account of AM perception at the neurophysiological, computational, and behavioral levels. Significance statementListening in everyday environments tasks the brain with extracting information from sound envelopes. This process involves both sensory encoding and decision-making. Different neural codes for envelope representation have been well characterized in the auditory midbrain and cortex, but studies of the brainstem have usually been conducted in anesthetized rodents or cats. Moreover, these candidate neural codes have been studied in isolation from the decision-making process. In this study, we found that population activity in the primate subcortical auditory system contains sufficient information for discriminating sound envelope and applied a biologically plausible model of decision-making to sound envelope discrimination performance from rhesus macaques, a species with great phylogenetic and perceptual similarity to humans.

neuroscience↗
bioRxiv2022

Metformin Improves Ovarian Function and Increases Egg Production in Broiler Breeder Hens

Broiler breeder hens, the parent stock of commercial broiler chickens, have poor reproductive efficiency associated with aberrant and excessive recruitment of ovarian follicles which results in sub-optimal egg production, fertility, and hatchability. The reproductive dysfunction observed in these hens resembles polycystic ovary syndrome in women, a condition wherein metformin is prescribed as a treatment. The main objectives of this study were to determine the effect of metformin on body weight, abdominal fat pad weight, ovarian function, and plasma steroid hormone concentrations. Broiler breeder hens were treated with 0, 25, 50, or 75 mg/kg body weight of metformin mixed in the diet for 40 weeks (n= 45 hens/treatment; 25-65 weeks of age). At 65 weeks of age, hens that received the highest dose of metformin had significantly lower body and abdominal fat pad weights (p < 0.05) than the control. Metformin treatment, at all levels, normalized the preovulatory and prehierarchical ovarian follicular hierarchy. Metformin (50 or 75 mg/kg body weight) significantly increased the total number of eggs laid per hen during the entire production period and these hens had significantly greater fertility and hatchability at 65 weeks of age compared to the control (p < 0.05). Metformin treatment at all levels altered the plasma profile of reproductive hormones, with significantly lower plasma testosterone concentrations and a decreased testosterone to androstenedione ratio in hens that received metformin (p < 0.05). Future studies should focus on the mechanisms underlying the beneficial effects of metformin in improving the reproductive efficiency of broiler breeder hens. In BriefThe pathophysiology of the ovarian dysfunction encountered in broiler breeder hens remains poorly understood but is similar to a condition in women known as PCOS. This study reveals that metformin may provide a cheap and effective method of improving ovarian function in broiler breeder hens.

physiology↗
bioRxiv2022

Correlations between behavior and neuronal responses in the cochlear nucleus (CN) and inferior colliculus (IC) of behaving monkeys

Detection of sounds is a fundamental function of the auditory system. While studies of auditory cortex have gained substantial insight into detection performance using behaving animals, previous subcortical studies have mostly taken place under anesthesia, in passively listening animals, or have not measured performance at threshold. These limitations preclude direct comparisons between neuronal responses and behavior. To address this, we simultaneously measured auditory detection performance and single-unit activity in the inferior colliculus (IC) and cochlear nucleus (CN) in macaques. The spontaneous activity and response variability of CN neurons were higher than those observed for IC neurons. Signal detection theoretic methods revealed that the magnitude of responses of IC neurons provided more reliable estimates of psychometric threshold and slope compared to the responses of single CN neurons. However, pooling small populations of CN neurons provided reliable estimates of psychometric threshold and slope, suggesting sufficient information in CN population activity. Trial-by-trial correlations between spike count and behavioral response emerged 50-75 ms after sound onset for most IC neurons, but for few neurons in the CN. These results highlight hierarchical differences between neurometric-psychometric correlations in CN and IC, and have important implications for how subcortical information could be decoded. New & NoteworthyThe cerebral cortex is widely recognized to play a role in sensory processing and decision-making. Accounts of the neural basis of auditory perception and its dysfunction are based on this idea. However, significantly less attention has been paid to midbrain and brainstem structures in this regard. Here we find that subcortical auditory neurons represent stimulus information sufficient for detection, and predict behavioral choice on a trial-by-trial basis.

neuroscience↗
bioRxiv2022

Conserved Pib2 regions have distinct roles in TORC1 regulation at the vacuole

TORC1 is a critical controller of cell growth in eukaryotes. In yeast, the presence of nutrients is signaled to TORC1 by several upstream regulatory sensors that together coordinate TORC1 activity. TORC1 localizes to both vacuolar and endosomal membranes, where differential signaling occurs. This localization is mimicked by Pib2, a key upstream TORC1 regulator that is essential for TORC1 reactivation after nutrient starvation or pharmacological inhibition. Pib2 has both positive and negative effects on TORC1 activity, but the mechanisms remain poorly understood. Here, we pinpoint the Pib2 inhibitory function on TORC1 to residues within short, conserved N-terminal regions. We also show that Pib2 C-terminal regions, helical region E and tail, are essential for TORC1 reactivation. Further, the Pib2 FYVE domain is essential for vacuolar localization, however, it is surprisingly unnecessary for recovery from rapamycin exposure. Using chimeric Pib2 targeting constructs, we show that endosomal localization is not necessary for TORC1 reactivation and cell growth after rapamycin treatment. Thus, a comprehensive molecular dissection of Pib2 demonstrates that each of its conserved regions differentially contribute to Pib2 regulation of TORC1 activity.

cell biology↗
bioRxiv2021

Temporal regulation of Pten is essential for retina regeneration in zebrafish

Unlike mammals, zebrafish possess a remarkable ability to regenerate damaged retina after an acute injury. Retina regeneration in zebrafish involves the induction of Muller glia-derived progenitor cells (MGPCs) exhibiting stem cell-like characteristics, which are capable of restoring all retinal cell-types. Here, we explored the importance of Phosphatase and tensin homolog (Pten), a dual-specificity phosphatase and tumor suppressor during retina regeneration. The Pten undergo rapid downregulation in the Muller glia and is absent in MGPCs, which is essential to trigger Akt-mediated cell proliferation to cause retina regeneration. We found that the forced downregulation of Pten accelerates MGPCs formation, while its overexpression restricts the regenerative response. We observed that Pten regulates the proliferation of MGPCs not only through Akt pathway but also by Mmp9/Notch signaling. Mmp9-activity is essential to induce the proliferation of MGPCs in the absence of Pten. Lastly, we show that Pten expression is fine-tuned through Mycb/histone deacetylase1 and Tgf-{beta} signaling. The present study emphasizes on the stringent regulation of Pten and its crucial involvement during the zebrafish retina regeneration.

cell biology↗
bioRxiv2021

Chrysomycin A inhibits the topoisomerase I of Mycobacterium tuberculosis.

Novel anti-tuberculosis drugs are essential to manage drug resistant tuberculosis, caused by the notorious pathogen Mycobacterium tuberculosis. We recently reported the antimycobacterial activity of chrysomycin A in vitro and in infected macrophages. In this study, we report that the molecule inhibits the growth of drug resistant clinical strains of Mycobacterium tuberculosis and acts in synergy with anti-TB drugs such as ethambutol, ciprofloxacin and novobiocin. In pursuit of its mechanism of action, it was found that chrysomycin A renders bactericidal activity by interacting with DNA at specific sequences and by inhibiting topoisomerase I activity of Mycobacterium tuberculosis. It also exhibits weak inhibition of gyrase enzyme of the pathogen.

biochemistry↗
bioRxiv2021

Assembly and levels of P-TEFb depend on reversiblephosphorylation of cyclinT1

The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, it is absent in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions require phosphorylation of two threonine residues (Thr143 and Thr149) in CycT1 by PKC. Conversely, PP1 dephosphorylates these sites. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminal differentiation of cells.

biochemistry↗
bioRxiv2021

Severe Acute Respiratory Syndrome Coronavirus-2 genome sequence variations relate to morbidity and mortality in Coronavirus Disease-19

Outcome of infection with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) may depend on the host, virus or the host-virus interaction-related factors. Complete SARS-CoV-2 genome was sequenced using Illumina and Nanopore platforms from naso-/oro-pharyngeal ribonucleic acid (RNA) specimens from COVID-19 patients of varying severity and outcomes, including patients with mild upper respiratory symptoms (n=35), severe disease ad-mitted to intensive care with respiratory and gastrointestinal symptoms (n=21), fatal COVID-19 outcome (n=17) and asymptomatic (n=42). Of a number of genome variants observed, p.16L>L (Nsp1), p.39C>C (Nsp3), p.57Q>H (ORF3a), p.71Y>Y (Membrane glycoprotein), p.194S>L (Nucleocapsid protein) were observed in similar frequencies in different patient subgroups. However, seventeen other variants were observed only in symptomatic patients with severe and fatal COVID-19. Out of the latter, one was in the 5UTR (g.241C>T), eight were synonymous (p.14V>V and p.92L>L in Nsp1 protein, p.226D>D, p.253V>V, and p.305N>N in Nsp3, p.34G>G and p.79C>C in Nsp10 protein, p.789Y>Y in Spike protein), and eight were non-synonymous (p.106P>S, p.157V>F and p.159A>V in Nsp2, p.1197S>R and p.1198T>K in Nsp3, p.97A>V in RdRp, p.614D>G in Spike protein, p.13P>L in nucleocapsid). These were completely absent in the asymptomatic group. SARS-CoV-2 genome variations have a significant impact on COVID-19 presentation, severity and outcome.

genomics↗
bioRxiv2021

Kinetic pathway of ATP-induced DNA interactions of ParA2, a protein essential for segregation of Vibrio cholerae chromosome 2

Vibrio cholerae chromosome 2 (Chr2) requires its own ParABS system for segregation. Without it, V. cholerae becomes nonviable and loses pathogenicity. ParA2 of Chr2 is a Walker-type ATPase that is the main driver of Chr2 segregation. Most of our understanding of ParA function comes from studying plasmid partition systems. How ParA provides the motive force in segregation of chromosomes, which are much larger than plasmids, is less understood and different models have been proposed. Here we analyzed in vivo behavior and kinetic properties of ParA2 using cell imaging, biochemical and biophysical approaches. ParA2 formed an asymmetric gradient in the cell that localized dynamically in the cell cycle. We found that ParA2 dimers bind ATP and undergo a slow conformational change to an active DNA-binding state, similar to P1 ParA. The presence of DNA catalyzes ParA2 conformational change to allow cooperative binding of active ParA2 dimers to form higher-order oligomers on DNA. Nucleotide exchange rates were also slow, thus providing a control of ParA2 recruitment and dynamic localizations. Although highly conserved in biochemical properties, ParA2 showed faster overall ATP cycling and DNA-rebinding rates than plasmid ParAs, suggesting that this could be shared kinetic features among chromosomal ParAs to regulate the transport of a much larger DNA cargo.

biochemistry↗
bioRxiv2021

S. aureus-serine protease-like protein B (SplB) activates PAR2 and induces endothelial barrier dysfunction

Withdrawal StatementThe authors have withdrawn their manuscript owing to the following reason: Unfortunately, the preparation of the recombinant serine protease-like protein B (SplB) used in this study was found to contain impurities, so that the effects presented cannot be unambiguously attributed to the protease SplB. We therefore retract the manuscript from bioRxiv. We apologize for any inconvenience this may have caused. Therefore, the authors do not wish this work to be cited as reference for the project. We want to clarify that the issue was identified in the preparation of the SplB protein and does not reflect on the contributions of individual authors. If you have any questions, please contact the corresponding author.

immunology↗