Biology subjects

Srinivasan, V.

Publications and source records attributed to Srinivasan, V..

14 recordsLinked to original sources

bioRxiv2026

An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

SHANK proteins form core postsynaptic density (PSD) scaffolds that organise synaptic signalling complexes through multiple protein-protein interaction domains, including PDZ domains that typically recognise C-terminal peptide motifs. Here, we identify an internal PDZ recognition mechanism that links the synaptic protein Densin-180 to SHANK and promotes SHANK scaffold assembly. We map SHANK binding to an internal PDZ-binding motif in Densin-180 (residues 843-863) and show by NMR spectroscopy and fluorescence polarisation that this motif binds SHANK1-3 PDZ domains with high affinity and specificity, competing with canonical C-terminal ligands. The crystal structure of the Densin-180-SHANK complex reveals that Phe858 inserts into the hydrophobic pocket of the PDZ domain despite the absence of a terminal carboxylate. In neurons, this interaction mediates Densin-180 recruitment to dendritic spines and drives activity-dependent reorganisation of postsynaptic SHANK3 assemblies, whereas mutation of Phe858 disrupts both processes. Disrupting the Densin-180-SHANK interaction using a Densin-180-derived peptide impairs activity-dependent structural plasticity of spines, accompanied by defects in PSD organisation and actin cytoskeleton remodelling. These findings define an internal mode of PDZ recognition and reveal how Densin-180 couples neuronal activity to the remodelling of postsynaptic SHANK scaffolds.

neuroscience↗

bioRxiv2025

Scalable prediction of symmetric protein complex structures

All life relies on proteins to function, yet accurately modeling protein structures that exceed {approx} 10, 000 amino acids or have higher-order geometries remains difficult. Existing solutions are limited to specific scenarios, require considerable computational resources, or are otherwise unscalable. Consequently, many large, disease-relevant protein complexes in the human proteome, as well as nearly all viruses and numerous other classes, are impractical to model with high fidelity for drug development. To modulate these protein complexes and viruses, structural information is eminently valuable, and often essential. In the last two years, machine learning based-tools that can generate binders to a given target structure with high hit rates have emerged. Combined with high-throughput screening, these technologies can far outpace traditional drug discovery. However, they cannot function well without accurate models of their target structures. Thus, to unlock the full power of AI-driven drug discovery, a scalable method must be developed to predict large protein complex structures. To overcome this bottleneck, we introduce Plica-1, a physics-based method to rapidly and accurately predict the structure of arbitrarily large, symmetric protein complexes. Validated across 4 major symmetry classes (icosahedral, tetrahedral, octahedral, and cyclic), the method consistently achieves near-experimental levels of accuracy, i.e., RMSD < 5[A]. In test cases, the method runs in < 5 minutes on consumer hardware, 103-105 times faster than the closest comparable software. The largest structure currently built, at {approx}40,000 amino acids, is > 8 times the limit of existing machine learning methods. The results demonstrate that protein complexes can be modeled at significantly improved speeds and scales, making Plica-1 a promising tool for protein engineering and drug development.

bioengineering↗

bioRxiv2025

Cyclin-dependent kinase-like 5 (CDKL5) binds to talin and is anchored at the postsynaptic density via direct interaction with PDZ domains

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine kinase essential for brain development and function. Mutations in the CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe early-onset epileptic encephalopathy characterised by defects in synapse formation and function. Despite extensive research, the molecular mechanisms by which CDKL5 mutations disrupt synaptic function and lead to epilepsy remain unclear. Here, we report that the major neuronal isoform of CDKL5 contains a C-terminal PDZ domain-binding motif. We demonstrate that this motif mediates interactions with the PDZ domains of PSD-95 and SHANK proteins, facilitating the recruitment of CDKL5 to the postsynaptic density. Disruption of CDKL5s PDZ-binding motif results in its mislocalisation and impaired spine formation. Additionally, we show that CDKL5 directly interacts with the mechanosensitive synaptic scaffold protein talin, via the N-terminal kinase domain of CDKL5 and the R8 rod domain of talin. Our findings establish how CDKL5 is targeted to synapses and suggest that its activity may be spatially regulated through talin-mediated mechanical signalling. We propose that the spatial positioning of the CDKL5 kinase domain might be mechanically-operated and regulated by talin domain unfolding. As talin undergoes structural transitions in its force-dependent binary switch domains, the kinase domain bound to R8 would be moved up and down within the synaptic compartment as a function of the changing talin conformation. These insights enhance our understanding of the pathogenic mechanisms underlying CDKL5 variants with premature stop codons and highlight the need to re-evaluate studies that have used C-terminally tagged or the non-PDZ-binding isoform of CDKL5 to assess its neuronal function. O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/672337v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@a11d72org.highwire.dtl.DTLVardef@19428b7org.highwire.dtl.DTLVardef@1fd1693org.highwire.dtl.DTLVardef@d5299c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Cyclin-dependent kinase-like 5 (CDKL5) is a mechanically regulated enzyme. The C-terminal PDZ-binding motif in CDKL5 anchors CDKL5 at the postsynaptic density (PSD). (A) The large unstructured region serves to tether the CDKL5 kinase domain at the synapse. i) The kinase domain is restricted to the blue semi-circle, giving CDKL5 a zone of activity, with radius the length of the tether (>200 nm). ii) Epilepsy-causing mutations that introduce premature stop codons untether CDKL5. (B) The mechanosensitive synaptic scaffold protein talin is also tethered to the membrane by interaction with integrins and/or amyloid precursor protein (APP). The CDKL5-binding domain, R8 is shown in dark green. The force-dependent binary switch domains in the talin rod region open and close in response to force and change the length of the molecule and the location of the CDKL5-binding site. (C) As talin changes its conformation, the CDKL5-binding site on R8 is moved up and down within the synapse, as a result the kinase activity of CDKL5 is targeted to discrete locations within the synapse. Loss of this localisation or incorrect positioning of the kinase within the synapse would result in loss of synchronisation of the synapse in the context of the circuit it is part of and, we propose, result in re-entry circuits and epilepsy. See also Supplementary Movie 1. C_FIG

neuroscience↗

bioRxiv2025

Increased proportion of growth-arrested bacilli in acidic pH adaptation promotes Mycobacterium tuberculosis treatment survival

The ability of Mycobacterium tuberculosis (Mtb) to adapt its growth behavior in response to host environments promotes survival against immune and drug stressors. However, how these behaviors shift at the single-cell level remains poorly understood. Here, we show that Mtb adapts to acidic conditions by increasing the proportion of bacteria in a growth-arrested state, rather than uniformly slowing the growth rate of the entire population. This non-growing subpopulation exhibits greater tolerance to ethambutol. Clinical strains displayed higher proportions of growth-arrested cells under both neutral and acidic conditions, suggesting that growth arrest serves as a bet-hedging strategy during infection. Though the PhoPR two-component system contributes to regulating this non-growing state, we show that it is a partial regulator of the non-growing bacterial subpopulation and that additional transcriptional regulators are involved. Our study demonstrates that non-growing subpopulations of Mtb provide fitness benefits and are an active adaptation to environmental cues and not a passive consequence of stressors.

microbiology↗

bioRxiv2024

USP14 regulates pS129 α-synuclein levels and oxidative stress in human SH-SY5Y dopaminergic cells

Ubiquitin specific protease-14 (USP14) is critical for controlling protein homeostasis disturbed in human disorders like Parkinso[n]s disease (PD). Here we investigated the role of USP14 in regulating proteasome and autophagy pathways, and their influence on -synuclein (-syn) degradation. Data showed that -syn and phosphorylated serine129 -syn (pS129 -syn) were elevated in USP14 gene-deleted SH-SY5Y dopaminergic cells with concomitant reduction in proteasome activity. Inhibition of proteasomes using MG132 particularly elevated pS129 -syn in these cells, but the levels were not influenced by inhibiting autophagy using chloroquine. In contrast, autophagy and the CLEAR (Coordinated Lysosomal Expression and Regulation) pathways were elevated in USP14 lacking cells with an upregulation of the transcription factor TFEB. USP14-ablated cells also exhibited increases in reactive oxidative species (ROS) and elongation of mitochondria. The addition of N-Acetylcysteine amide (NACA) to counteract oxidative stress, reduced pS129 -syn and -syn levels in USP14 deficient cells. Phospho-proteomic analyses revealed that USP14 is phosphorylated at S143 affecting its function and structure as shown by molecular modeling, and protein interaction studies. Re-expression of wild-type and the phospho-mimetic S143D-USP14 mutant decreased ROS, pS129 -syn, and -syn in USP14 lacking cells. These results demonstrate that pS129 -syn levels are sensitive to oxidative stress in SH-SY5Y dopaminergic cells. USP14 by stimulating the proteasome activity and reducing oxidative stress is a promising factor for targeting -syn and its pathogenic variants in PD.

neuroscience↗

bioRxiv2023

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. We determined the in-vitro rifampicin survival fraction by minimum duration of killing assay in isoniazid susceptible (IS, n=119) and resistant (IR, n=84) M. tuberculosis isolates. Then we correlated the rifampicin tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal IR isolates collected from patients were analyzed for changes in rifampicin tolerance and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation. This indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log10-fold survival fraction enabled classification of tolerance as low, medium or high and revealed IR association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P-trend=0.0003). The high tolerance in IR isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Furthermore, the high rifampicin tolerant IR isolates have survival potential similar to multi-drug resistant isolates. These findings suggest that IR tuberculosis needs to be evaluated for high rifampicin tolerance to improve treatment regimen and prevent the risk of MDR-TB emergence.

microbiology↗

bioRxiv2023

Nucleoporin-binding nanobodies that either track or inhibit nuclear pore complex assembly

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact NPCs from frog to human. We further report a simplified assay that directly tracks postmitotic NPC assembly by added labeled anti-Nup nanobodies. In interphase, NPCs are inserted into a pre-existing nuclear envelope. This makes it difficult to monitor this process as newly-assembled NPCs must be distinguished from pre-existing ones. We solved this problem by inserting Xenopus NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We also asked whether anti-nucleoporin (Nup) nanobodies could serve as NPC assembly inhibitors. A first generation, selected from immune libraries against Xenopus Nups, comprised only bright stainers of intact NPCs but no inhibitors, perhaps because the immune response was biased towards non-conserved and, thus, functionally-irrelevant epitopes. To overcome this bias, we selected for crossreactivity between Xenopus and human Nups and obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93--solenoid in recruiting Nup358 and the Nup214{middle dot}88{middle dot}62 complex, and for Nup155 and the Nup98 autoproteolytic domain in NPC-scaffold assembly. The latter suggests an assembly checkpoint linking pore formation to permeability barrier assembly.

cell biology↗

bioRxiv2023

Revisiting the role of Acinetobacter sp. in EBPR systems

Side-stream Enhanced biological phosphorus removal (S2EBPR) has been incorporated with B-stage process to enable simultaneous phosphorus and nitrogen removal. However, the dominating phosphorus accumulating organisms (PAOs) in this novel configuration has not been evaluated. The dominance of Acinetobacter was confirmed by 16S sequencing. In addition, single cell Raman spectrum (SCRS) analysis in couple with in situ fluorescence in situ hybridization (FISH) was applied to obtain the feature spectrum and verify the phosphorus release/uptake activity of Acinetobacter spp. The phenotypic profiling further suggested the dominance of Acinetobacter-like organisms among all poly-phosphorus containing organisms and only certain phenotypic Acinetobacter (oligotype 1) contribution to P-removal in a unique HRAS-P(D)N-S2EBPR system. The findings suggest that Acinetobacter may outcompete other heterotrophic organisms in EBPR systems due to their sensitivity to operational conditions. However, stable P-removal was only observed during a specific section of the operation period, coinciding with an increase in the VFA/P ratio. Further research is needed to identify the phenotypes of Acinetobacter responsible for P-removal in EBPR systems. The study contributes to a better understanding of the microbial ecology and engineering aspects of EBPR systems and wastewater treatment in general.

bioengineering↗

bioRxiv2023

Molecular Evidence of Internal Carbon-Driven Partial Denitrification Annamox (PdNA) in a mainstream Pilot A-B System Coupled with Side-stream EBPR treating municipal wastewater

Achieving mainstream short-cut nitrogen removal via nitrite has been a carbon and energy-efficient goal which wastewater engineers are dedicated to explore. This study applied a novel pilot-scale A-B-S2EBPR system process integrated with sidestream enhanced biological phosphorus removal) to achieve the nitrite accumulation and downstream anammox for treating municipal wastewater. Nitrite accumulated to 5.5 {+/-} 0.3 mg N/L in the intermittently aerated tanks of B-stage with the nitrite accumulation ratio (NAR) of 79.1 {+/-} 6.5%. The final effluent concentration and removal efficiency of total inorganic nitrogen (TIN) were 4.6 {+/-} 1.8 mg N/L and 84.9 {+/-} 5.6%, respectively. Batch nitrification/denitrification activity tests and functional gene abundance of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) suggested that the nitrite accumulation was mostly caused by partial denitrification without NOB- selection. The unique features of S2EBPR (longer anaerobic HRT/SRT, lower ORPs, high and more complex VFAs etc.) seemed to impact the nitrogen microbial communities: the conventional AOB kept at a very low level of 0.13 {+/-} 0.13% during the operation period, and the dominant candidate internal carbon-accumulating heterotrophic genera of Acinetobacter (17.8 {+/-} 15.5)% and Comamonadaceae (6.7 {+/-} 3.4%) were highly enriched. Furthermore, the single-cell Raman spectroscopy-based intracellular polymer analysis revealed the dominate microorganisms that could utilize polyhydroxyalkanoates (PHA) as the potential internal carbon source to drive partial denitrification. This study provides insights and a new direction for implementing the mainstream PdNA short-cut nitrogen removal via incorporating S2EBPR into sustainable A-B process.

bioengineering↗

bioRxiv2021

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗

bioRxiv2021

Integrative COVID-19 Biological Network Inference with Probabilistic Core Decomposition

The SARS-CoV-2 coronavirus is responsible for millions of deaths around the world. To help contribute to the understanding of crucial knowledge and to further generate new hypotheses relevant to SARS-CoV-2 and human protein interactions, we make use of the information abundant Biomine probabilistic database and extend the experimentally identified SARS-CoV-2-human protein-protein interaction (PPI) network in silico. We generate an extended network by integrating information from the Biomine database, the PPI network, and other experimentally validated results. To generate novel hypotheses, we focus on the high-connectivity sub-communities that overlap most with the integrated experimentally validated results in the extended network. Therefore, we propose a new data analysis pipeline that can efficiently compute core decomposition on the extended network and identify dense subgraphs. We then evaluate the identified dense subgraph and the generated hypotheses in three contexts: literature validation for uncovered virus targeting genes and proteins, gene function enrichment analysis on subgraphs, and literature support on drug repurposing for identified tissues and diseases related to COVID-19. The majority types of the generated hypotheses are proteins with their encoding genes and we rank them by sorting their connections to the integrated experimentally validated nodes. In addition, we compile a comprehensive list of novel genes, and proteins potentially related to COVID-19, as well as novel diseases which might be comorbidities. Together with the generated hypotheses, our results provide novel knowledge relevant to COVID-19 for further validation.

bioinformatics↗

bioRxiv2020

Machine Leaning-based Determination of Sampling Depth for Complex Environmental Systems: Case Study with Single-Cell Raman Spectroscopy Data in EBPR Systems

Rapid progress in various advanced analytical methods such as single-cell technologies enable unprecedented and deeper understanding of microbial ecology beyond the resolution of conventional approaches. A major application challenge exists in the determination of sufficient sample size without sufficient prior knowledge of the community complexity and, the need to balance between statistical power and limited time or resources. This hinders the desired standardization and wider application of these technologies. Here, we proposed, tested and validated a computational sampling size assessment protocol taking advantage of a metric, named kernel divergence. This metric has two advantages: First, it directly compares dataset-wise distributional differences with no requirements on human intervention or prior knowledge-based pre-classification. Second, minimal assumptions in distribution and sample space are made in data processing to enhance its application domain. This enables test-verified appropriate handling of datasets with both linear and non-linear relationships. The model was then validated in a case study with eight SCRS phenotyping datasets each sampled from a different enhanced biological phosphorus removal (EBPR) activated sludge community located across North America. The model allows the determination of sufficient sampling size for any targeted or customized information capture capacity or resolution level. For example, an approximated sampling size of 50 or 100 spectra for full-scale EBPR-related ecosystems at 5% or 2% OPU cluster resolution. Promised by its flexibility and minimal restriction of input data types, the proposed method is expected to be a standardized approach for sampling size optimization, enabling more comparable and reproducible experiments and analysis on complex environmental samples. Finally, these advantages exhibit the capability of generalizing to other single-cell technologies or environmental applications, provided that the input datasets contain only continuous features. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/423496v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1261a6eorg.highwire.dtl.DTLVardef@91369aorg.highwire.dtl.DTLVardef@197f64dorg.highwire.dtl.DTLVardef@1ab3c19_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2020

Modeling Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) System Using Agent-based Model with Adaptive Maintenance, Decay and TCA Metabolism

Side-stream enhanced biological phosphorus removal process (S2EBPR) has been demonstrated to improve performance stability and offer a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR require modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved model (iEBPR) for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay, and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against bulk batch testing experiment data, being proved to perform better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen, and PHA temporal profiles in a batch starvation testing under prolonged anaerobic conditions. We further validated the model with another independent set of batch anaerobic batch testing data that included high-resolution single-cell and specific population level intracellular polymer measurements enabled by the single-cell Raman micro-spectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, and further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance-decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantages over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.

microbiology↗

bioRxiv2020

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

biophysics↗