Biology subjects

Das, R.

Publications and source records attributed to Das, R..

At least 127 records · Page 7Linked to original sources

bioRxiv2020

Rational design of protein-specific folding modifiers

Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a xenonucleus, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Forster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 {+/-} 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

biophysics↗

bioRxiv2020

De novo 3D models of SARS-CoV-2 RNA elements and small-molecule-binding RNAs to guide drug discovery

The rapid spread of COVID-19 is motivating development of antivirals targeting conserved SARS-CoV-2 molecular machinery. The SARS-CoV-2 genome includes conserved RNA elements that offer potential small-molecule drug targets, but most of their 3D structures have not been experimentally characterized. Here, we provide a compilation of chemical mapping data from our and other labs, secondary structure models, and 3D model ensembles based on Rosettas FARFAR2 algorithm for SARS-CoV-2 RNA regions including the individual stems SL1-8 in the extended 5 UTR; the reverse complement of the 5 UTR SL1-4; the frameshift stimulating element (FSE); and the extended pseudoknot, hypervariable region, and s2m of the 3 UTR. For eleven of these elements (the stems in SL1-8, reverse complement of SL1-4, FSE, s2m, and 3 UTR pseudoknot), modeling convergence supports the accuracy of predicted low energy states; subsequent cryo-EM characterization of the FSE confirms modeling accuracy. To aid efforts to discover small molecule RNA binders guided by computational models, we provide a second set of similarly prepared models for RNA riboswitches that bind small molecules. Both datasets ( FARFAR2-SARS-CoV-2, https://github.com/DasLab/FARFAR2-SARS-CoV-2; and FARFAR2-Apo-Riboswitch, at https://github.com/DasLab/FARFAR2-Apo-Riboswitch) include up to 400 models for each RNA element, which may facilitate drug discovery approaches targeting dynamic ensembles of RNA molecules.

biochemistry↗

bioRxiv2020

Genetically encoded live cell sensor for tyrosinated microtubules

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin post-translational modification (PTMs). These PTMs are known to affect microtubule stability, dynamics and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present there exist no tool that can specifically mark tubulin PTMs in live cells, thus severely limiting our understanding of tubulin PTMs. Using yeast display library, we identified a binder against terminal tyrosine of alpha tubulin, a unique PTM site. Extensive characterization validates the robustness and non-perturbing nature of our binder as tyrosination sensor, a live cell tubulin nanobody specific towards tyrosinated or unmodified microtubules. Using which, in real time we followed nocodazole, colchicine and vincristine induced depolymerization events of unmodified microtubules, and found each distinctly perturb microtubule polymer. Together, our work describes the tyrosination sensor and potential applications to study microtubule and PTM processes in living cells.

cell biology↗

bioRxiv2020

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences reported to date from the current COVID-19 outbreak, and we present a curated list of 30 SARS-related-conserved regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 SARS-CoV-2-conserved-structured regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the 5 UTR, frame-shifting element, and 3 UTR. Last, we predict regions of the SARS-CoV-2 viral genome have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 SARS-CoV-2-conserved-unstructured genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

genetics↗

bioRxiv2020

Accelerating Maximal-Exact-Match Seeding with Enumerated Radix Trees

MotivationRead alignment is a time-consuming step in genome sequence analysis. In the read alignment software BWA-MEM and the recently published faster version BWA-MEM2, the seeding step is a major bottleneck, for instance, contributing 38% to the overall execution time in BWA-MEM2 when aligning single-end whole human genome reads from the Platinum Genomes dataset. This is because both BWA-MEM and BWA-MEM2 use a compressed index structure called the FMD-Index, which results in high memory bandwidth requirements for seeding, primarily due to its character-by-character processing of reads. ResultsWe propose a memory bandwidth-aware data structure for maximal-exact-match seeding called Enumerated Radix Tree (ERT). ERT trades off memory capacity to improve seeding performance ([~]60 GB index for human genome). Together with optimizations to the seeding algorithm and mate-rescue step, ERT when integrated into BWA-MEM2 speeds up overall read alignment by 1.28x and provides up to 2.1x higher seeding performance while guaranteeing identical output to the original software. Furthermore, we prototype an FPGA implementation of ERT on Amazon EC2 F1 cloud and observe 1.6x higher seeding throughput over a 48-thread optimized CPU-ERT implementation. Availability and implementationhttps://github.com/arun-sub/bwa-mem2 Contactarunsub@umich.edu, reetudas@umich.edu

bioinformatics↗

bioRxiv2020

In silico chemical profiling and identification of neuromodulators from Curcuma amada targeting Acetylcholinesterase

Curcuma amada or Mango ginger, a member of the Zingiberaceae family, has been revealed as a beneficiary medicinal plant having diverse pharmacological activities against a wide range of diseases. Due to having neuromodulation properties of this plant, the present study characterized the secondary metabolites of Curcuma amada for their drug-likeness properties, identified potent hits by targeting Acetylcholinesterase (AChE) and revealed neuromodulatory potentiality by network pharmacology approaches. Here in silico ADMET analysis was performed for chemical profiling, and molecular docking and molecular dynamics simulations were used to hit selection and binding characterizations. Accordingly, ADMET prediction showed that around 87.59% of compounds processed drug-likeness activity, where four compounds have been screened out by molecular docking. Guided from induced-fit docking, molecular dynamics simulations revealed phytosterol and curcumin derivatives as the most favorable AChE inhibitors with the highest binding energy, as resulted from MM-PBSA analysis. Furthermore, all of the four hits were appeared to modulate several signaling molecules and intrinsic cellular pathways in network pharmacology analysis, which are associated with neuronal growth survival, inflammation, and immune response, supporting their capacity to revert the condition of neuro-pathobiology. Together, the present in silico based characterization and system pharmacology based findings demonstrate Curcuma amada, as a great source of neuromodulating compounds, which brings about new development for complementary and alternative medicine for the prevention and treatment of neurodegenerative disorders.

bioinformatics↗

bioRxiv2020

Anomalous reverse transcription through chemical modifications in polyadenosine stretches

Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2 hydroxyl acylation, as probed by reverse transcription. Here, re-analysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on A12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3 untranslated region RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.

biochemistry↗

bioRxiv2019

Crowdsourced RNA design discovers diverse, reversible, efficient, self-contained molecular sensors

Internet-based scientific communities promise a means to apply distributed, diverse human intelligence towards previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale videogame-based crowdsourcing of functional RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near-thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs--results that surpass computational and expert-based design. This work represents a new paradigm for widely distributed experimental bioscience. One Sentence SummaryOnline community discovers standalone RNA sensors.

synthetic biology↗

bioRxiv2019

Folding heterogeneity in the essential human telomerase RNA three-way junction

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (3WJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, endogenous RNP assembly assays, and single-molecule structural analysis. Our data suggest that a functionally essential stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase protein subunit in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. RNA structural engineering intended to bias the folding landscape of the hTR CR4/5 demonstrates that a stably folded 3WJ motif is necessary but not sufficient to promote assembly of a functional RNP complex. Finally, single-molecule measurements on the hTR CR4/5 domain show that RNP assembly selects for a conformation that is not the major population in the heterogeneous free RNA ensemble, suggesting that non-canonical hTR folds may be required during telomerase biogenesis.

biochemistry↗

bioRxiv2019

Melatonin reduces GSK3β expression and tau phosphorylation via Nrf2 nuclear translocation

Alzheimers disease is a neuropathological condition with abnormal formation of extracellular Amyloid-{beta} plaques and intracellular neurofibrillary tangles (NFTs) of Microtubule-associated protein Tau (Tau) in brain. In pathological condition, MAP-Tau can undergo various post-translational modifications such as hyperphosphorylation by the activity of cellular kinases which eventually leads to protein aggregation in neurons. Melatonin is a hormone which mainly secreted from pineal gland, functions to modulate cellular kinases. In our study, we elucidated that Melatonin has inhibited the Tau aggregates mediated cytotoxicity and membrane leakage by MTT and LDH assay respectively in neuro2A cells. Melatonin has found to reduce the GSK3{beta} mRNA expression and protein level by western blot and immunofluorescence assay. Melatonin has also decreased phospho-Tau level (pThr181 and pThr212-pSer214) in neuron cell line upon OA induction as seen by microscopic analysis.. Melatonin treatment has associated with ROS quenching by DCFDA assay, reduced caspase 3 activity in neuronal cells. Further, Melatonin has increased Nrf2 level and nuclear translocation as oxidative stress response in Tauopathy. Together, these findings clearly signifies that Melatonin remediate the Tau-induced neuronal cytotoxicity and reduce Tau hyperphosphorylation via downregulating GSK3{beta} expression. Melatonin can combat oxidative damage by Nrf2 activation and nuclear translocation in AD condition.

cell biology↗

bioRxiv2019

Effect of Melatonin on Tau aggregation and Tau-mediated cell surface morphology

Tau is the major neuronal protein involved in the stabilization of microtubule assembly. In Alzheimers disease, Tau self assembles to form intracellular protein aggregates, which are toxic to cells. Various methods have been tried and tested to restrain the aggregation of Tau. Most of the agents tested for this purpose have limitations in their effectiveness and availability to neuronal cells. We tested melatonin against in vitro Tau aggregation and observed its effect on membrane topology, tubulin network and Tau phosphorylation in neuro2a and N9 cell lines. The aggregation and conformation of Tau was determined by ThT fluorescence and CD spectroscopy respectively. The morphology of Tau aggregates in presence and absence of melatonin was studied by transmission electron microscopy. Melatonin was found to reduce the formation of higher order oligomeric structures without affecting the overall aggregation kinetics of Tau. Melatonin also modulates and helps to maintain membrane topology as evidenced by FE-SEM analysis. Overall, melatonin administration shows mild anti-aggregation and cytoprotective effects.

cell biology↗

bioRxiv2019

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

{beta}-glucosidase catalyzes the hydrolysis of {beta}-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of {beta}-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with {beta}-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a {beta}-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the {beta}-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of {beta}-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

biochemistry↗

bioRxiv2019

An allosteric hot-spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling

T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly-phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-phosphorylated ITAM-{zeta}1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells. SignificanceT-cell and B-cell signaling is initiated by the same family of non-receptor tyrosine kinases, ZAP-70 and Syk, respectively. ZAP-70 and Syk share homologous sequence and similar structural architecture, yet the two kinases differ in their mode of ligand recognition. ZAP-70 binds cooperatively to its ligand, whereas Syk binds uncooperatively. Spontaneous mutation (W165C) in the regulatory module of ZAP-70 impairs T-cell signaling causes autoimmune arthritis in SKG mice, the mechanism of which is unknown. We showed that ZAP-70 regulatory module undergoes a biphasic structural transition while binding to its ligand, which is fundamentally different from Syk. We presented a molecular mechanism of cooperativity in ZAP-70 regulatory module that explains altered ligand binding by ZAP-70 mutant found in SKG mice.

biochemistry↗

bioRxiv2019

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR enzymes occurs in double-stranded RNAs (dsRNAs). How the RNA sequence and structure (i.e., the cis-regulation) determine the editing efficiency and specificity is poorly understood, despite a compelling need towards functional understanding of known editing events and transcriptome engineering of desired adenosines. We developed a CRISPR/Cas9-mediated saturation mutagenesis approach to generate comprehensive libraries of point mutations near an editing site and its editing complementary sequence (ECS) at the endogenous genomic locus. We used machine learning to integrate diverse RNA sequence features and computationally predicted structures to model editing levels measured by deep sequencing and identified cis-regulatory features of RNA editing. As proof-of-concept, we applied this integrative approach to three editing substrates. Our models explained over 70% of variation in editing levels. The models indicate that RNA sequence and structure features synergistically determine the editing levels. Our integrative approach can be broadly applied to any editing site towards the goal of deciphering the RNA editing code. It also provides guidance for designing and screening of antisense RNA sequences that form dsRNA duplex with the target transcript for ADAR-mediated transcriptome engineering.

molecular biology↗

bioRxiv2019

Cortical anchoring of the microtubule cytoskeleton is essential for neuron polarity and functioning

Neurons are among the most highly polarized cell types. They possess structurally and functionally different processes, axon and dendrites, to mediate information flow through the nervous system. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is little understood.\n\nUsing the in vivo model system Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cell cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.

cell biology↗

bioRxiv2019

MicroRNA-194 promotes lineage plasticity in advanced prostate cancer

MicroRNA-194 (miR-194) promotes prostate cancer metastasis, but the precise molecular mechanisms by which it achieves this are unknown. Here, by integrating Argonaute high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (Ago-HITS-CLIP) with RNA sequencing and exon-intron split analysis, we defined a 163-gene miR-194 \"targetome\" in prostate cancer. These target genes were predominantly down-regulated through canonical 3UTR recognition sites and were enriched within pathways involved in cytoskeletal organisation and cell movement. In clinical prostate cancer samples, miR-194 activity was inversely correlated with the androgen receptor (AR) signalling axis. At a mechanistic level, this inverse correlation was explained by down-regulation of miR-194 expression by AR. Accordingly, miR-194 expression and activity was significantly elevated in neuroendocrine prostate cancer (NEPC), an aggressive AR-independent disease subtype. MiR-194 enhanced the transdifferentiation of prostate adenocarcinoma cells to a neuroendocrine-like state, at least in part by targeting FOXA1, a transcription factor with a key role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor effectively inhibited the growth of cell lines and patient-derived organoids with neuroendocrine features. Overall, our study reveals a novel post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in this NEPC.

cancer biology↗

bioRxiv2019

FARFAR2: Improved de novo Rosetta prediction of complex global RNA folds

Methods to predict RNA 3D structures from sequence are needed to understand the exploding number of RNA molecules being discovered across biology. As assessed during community-wide RNA-Puzzles trials, Rosettas Fragment Assembly of RNA with Full-Atom Refinement (FARFAR) enables accurate prediction of complex folds, but it remains unclear how much human intervention and experimental guidance is needed to achieve this performance. Here, we present FARFAR2, a protocol integrating recent innovations with updated RNA fragment libraries and helix modeling. In 16 of 21 RNA-Puzzles revisited without experimental data or expert intervention, FARFAR2 recovers structures that are more accurate than the original models submitted by our group and other participants during the RNA-Puzzles trials. In five prospective tests, pre-registered FARFAR2 models for riboswitches and adenovirus VA-I achieved 3-8 [A] RMSD accuracies. Finally, we present a server and three large model archives (FARFAR2-Classics, FARFAR2-Motifs, and FARFAR2-Puzzles) to guide future applications and advances.

biochemistry↗

bioRxiv2019

A novel polyubiquitin chain linkage formed by viral Ubiquitin prevents cleavage by deubiquitinating enzymes

The Baculoviridae family of viruses encode a viral Ubiquitin gene. Although the viral Ubiquitin is homologous to eukaryotic Ubiquitin (Ub), preservation of this gene in the viral genome indicates a unique function that is absent in the host eukaryotic Ub. We report the structural, biophysical, and biochemical properties of the viral Ubiquitin from Autographa Californica Multiple Nucleo-Polyhedrosis Virus (AcMNPV). The structure of viral Ubiquitin (vUb) differs from Ub in the packing of the central helix 1 to the beta-sheet of the {beta}-grasp fold. Consequently, the stability of the fold is lower in vUb compared to Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54). The K54-linked polyubiquitin chains are neither effectively cleaved by deubiquitinating enzymes, nor are they targeted by proteasomal degradation. We propose that modification of proteins with the viral Ubiquitin is a mechanism to counter the host antiviral responses.

biochemistry↗