Biology subjects

Chowdhury, S.

Publications and source records attributed to Chowdhury, S..

108 records · Page 6Linked to original sources

bioRxiv2020

A guided approach for subtomogram averaging of challenging macromolecular assemblies

Cryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

molecular biology↗

bioRxiv2020

Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2

Tight regulatory mechanisms to maintain repression of human Telomerase (hTERT), the sole protein that synthesizes telomeres, is crucial for normal adult somatic cells. In contrast, enhanced telomerase activity and resulting pathological maintenance of telomeres, is widely understood as causal in >90% of human cancers. These implicate underlying mechanisms connecting hTERT regulation and telomeres, possibly through telomeric proteins, that remain unclear. In light of of recent work by us and others showing non-telomeric function of the telomere-binding protein TRF2, here we examined whether and how TRF2 affected hTERT regulation. Direct binding of TRF2 - spanning [~]450 bp of the hTERT promoter from the Transcriptional Start Site (TSS) - led to TRF2-dependent recruitment of the polycomb repressor complex PRC2 in both normal and cancer cells. This induced repressor histone modifications resulting in TRF2-dependent hTERT repression. Mutations in the hTERT promoter, found frequently in aggressive glioblastoma and reported to destabilize the G-quadruplex structure, resulted in loss of TRF2 binding and consequent hTERT over-expression. Conversely, using G-quadruplex-stabilizing ligands we regained TRF2 binding, hTERT re-suppression, in highly proliferating glioblastoma cells with telomerase hyperactivation due to hTERT promoter mutations. Together, results herein demonstrate direct control of hTERT through TRF2 in a G-quadruplex-dependent manner - implicating mechanisms of how telomerase regulation might be linked to telomeres in normal and cancer cells.

cancer biology↗

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

Chimeric LuxR Transcription Factors Rewire Natural Product Regulation

LuxR-type transcriptional activator proteins frequently flank bacterial biosynthetic gene clusters (BGCs) where they play a crucial role in regulating natural product formation. Only few bacterial BGCs are expressed under standard culturing conditions, thus modulation of flanking LuxRs is a powerful approach to activate silent clusters. Here, we show that exploiting the modular nature LuxR proteins and constructing chimeric LuxRs enables the activation of BGCs.

microbiology↗

bioRxiv2019

Programmable bacteria induce durable tumor regression and systemic antitumor immunity

SUMMARY PARAGRAPHSynthetic biology is driving a new era of medicine through the genetic programming of living cells1,2. This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics3-5. One particular focus area has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo6-8. Here, we engineered a non-pathogenic E. coli to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb)9, an anti-phagocytic receptor commonly overexpressed in several human cancers10,11. We show that intratumoral delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Moreover, we report that local injection of CD47nb bacteria stimulates systemic antitumor immune responses that reduce the growth of untreated tumors - providing, to the best of our knowledge, the first demonstration of an abscopal effect induced by a bacteria cancer therapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

synthetic biology↗

bioRxiv2019

Crosslinking Proteomics Indicates Effects of Simvastatin on the TLR2 interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade

Toll-like receptor 2 (TLR2) is a pattern recognition receptor that, upon ligation by microbial molecules, interacts with other proteins to initiate pro-inflammatory responses by the cell. Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors), drugs widely prescribed to reduce hypercholesterolemia, are reported to have both pro- and anti-inflammatory effects upon cells. Some of these responses are presumed to be driven by effects on signaling proteins at the plasma membrane, but the underlying mechanisms remain obscure. We reasoned that profiling the effect of statins on the repertoire of TLR2-interacting proteins might provide novel insights into the mechanisms by which statins impact inflammation. In order to study the TLR2 interactome, we designed a co-immunoprecipitation (IP)-based cross-linking proteomics study. A hemagglutinin (HA)-tagged-TLR2 transfected HEK293 cell line was utilized to precipitate the TLR2 interactome upon cell exposure to the TLR2 agonist Pam3CSK4 and simvastatin, singly and in combination. To stabilize protein interactors, we utilized two different chemical cross-linkers with different spacer chain lengths. Proteomic analysis revealed important combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome. After stringent data filtering, we identified alpha-centractin (ACTR1A), an actin-related protein and subunit of the dynactin complex, as a potential interactor of TLR2. The interaction was validated using biochemical methods. RNA interference studies revealed an important role for ACTR1A in induction of pro-inflammatory cytokines. Taken together, we report that statins remodel the TLR2 interactome, and we identify ACTR1A, a part of the dynactin complex, as a novel regulator of TLR2-mediated immune signaling pathways.

immunology↗

bioRxiv2019

Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including zebrafish Danio rerio, we identified two populations of white pigment cells--leucophores--one of which arises by transdifferentiation of adult melanophores and another that develops from a yellow/orange xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical and ultrastructural analyses of zebrafish leucophores revealed cell-type specific chemical compositions, organelle configurations and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching and we show that leucophore complement influences behavior. Together, our studies revealed new, independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish, and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

developmental biology↗

bioRxiv2019

Dissociating orexin-dependent and -independent functions of orexin neurons using novel orexin-Flp knock-in mice

Uninterrupted arousal is important for survival during threatening situations. Activation of orexin/hypocretin neurons is implicated in sustained arousal. However, orexin neurons produce and release orexin as well as several co-transmitters including dynorphin and glutamate. Thus, it is important to disambiguate orexin peptide-dependent and -independent physiological functions of orexin neurons. To attain this, we generated a novel orexin-flippase (Flp) knock-in (KI) mouse line. Crossing with Flp-reporter or Cre-expressing mice showed gene expression exclusively in orexin neurons. Histological studies confirmed that orexin was completely knock-out (KO) in KI/KI homozygous mice. Orexin neurons without orexin showed altered electrophysiological properties, as well as received decreased glutamatergic inputs. Selective chemogenetic activation revealed that both orexin and co-transmitters functioned to increase wakefulness, however, orexin was indispensable to promote sustained arousal. Surprisingly, activation of orexin neurons without orexin caused a significant increase in the total time spent in cataplexy. Taken together, orexin is essential to maintain basic membrane properties and input-output computation of orexin neurons, as well as to exert awake-sustaining aptitude of orexin neurons.

neuroscience↗

bioRxiv2019

GABA neurons in the ventral tegmental area regulate non-rapid eye movement sleep in mice

The daily sleep/wakefulness cycle is regulated by coordinated interactions between sleep- and wakefulness-regulating neural circuitry. However, the detailed neural circuitry mediating sleep is far from understood. Here, we found that glutamic acid decarboxylase 67 (Gad67)-positive GABAergic neurons in the ventral tegmental area (VTAGad67+) are a key regulator of non-rapid eye movement (NREM) sleep in mice. VTAGad67+ neurons project to multiple brain areas implicated in sleep/wakefulness regulation such as the lateral hypothalamus (LH) and dorsal raphe nucleus. Chemogenetic activation of VTAGad67+ neurons promoted NREM sleep with higher delta power whereas optogenetic inhibition of these neurons induced prompt arousal from NREM sleep under highly somnolescent conditions, but not during REM sleep. In vivo fiber photometry recordings revealed that VTAGad67+ neurons showed the highest population activity in NREM sleep and the lowest activity in REM sleep. Acute brain slice electrophysiology combined with optogenetics revealed that VTAGad67+ neurons directly innervate and inhibit wake-promoting orexin/hypocretin neurons in the LH by releasing GABA. Taken together, we reveal that VTAGad67+ neurons play a crucial role in the regulation of NREM sleep.

neuroscience↗

bioRxiv2018

Fibroblast activation protein enzyme deficiency prevents liver steatosis, insulin resistance and glucose intolerance and increases fibroblast growth factor-21 in diet induced obese mice

Background & AimsFibroblast activation protein-a (FAP) is a post-proline peptidase closely related to dipeptidyl peptidase-4. FAP degrades bioactive peptides including fibroblast growth factor-21 (FGF-21) and neuropeptide Y. We examined metabolic outcomes of specific genetic ablation of FAP and its enzyme activity in a mouse model of diet-induced obesity (DIO) causing fatty liver.\n\nMethodsWildtype (WT) and genetically modified FAP deficient mice that specifically lacked either the FAP protein or FAP enzyme activity received chow, or an atherogenic diet for 8 to 20 weeks of DIO.\n\nResultsFAP deficient male and female mice in the DIO model were more metabolically healthy than controls. The FAP deficient mice had less glucose intolerance, liver lipid, adiposity, insulin resistance, pancreatic and plasma insulin, pancreatic {beta}-cell hyperplasia, serum alanine transaminase and circulating cholesterol compared to wild type controls. Furthermore, FAP deficiency lowered respiratory exchange ratio and greatly increased intrahepatic non-esterified free fatty acids, indicative of increased lipolysis and {beta}-oxidation. Concordantly, lipogenic genes (Pparg, Gck, Acc, Fasn) and hepatic triglyceride and fatty acid uptake genes (Cd36, Apoc3, Ldlr) and plasma low-density lipoprotein cholesterol were downregulated. Glucagon like peptide-1 levels were unaltered. FAP was localized to human pancreatic {beta}-cells and pancreas from diabetes mellitus patients contained elevated FAP activity. Comparable data from a FAP gene knockout mouse and a novel mouse lacking FAP enzyme activity indicated that these metabolic changes depended upon the enzymatic activity of FAP. These changes may be driven by FGF-21, which was upregulated in livers of FAP deficient DIO mice.\n\nConclusionThis is the first study to show that specific genetic ablation of FAP activity or protein protects against DIO-driven glucose intolerance, hyperinsulinaemia, insulin resistance, hypercholesterolaemia and liver steatosis in mice and provide mechanistic insights.

pathology↗

bioRxiv2018

Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here we present the cryo-EM structure of the type I-F CRISPR RNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a I180-degree rotation of the C-terminal helical bundle on the \"large\" Cas8f subunit. We show that the dsDNA-induced conformational change in Cas8f exposes a Cas2/3 \"nuclease recruitment helix\" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f \"nuclease recruitment helix\", implying that cas genes may sometimes serve as genetic fodder for the evolution of anti-CRISPRs.

molecular biology↗

bioRxiv2018

Mechanisms for zinc and proton inhibition of the GluN1/GluN2A NMDA receptor

N-Methyl-D-aspartate receptors (NMDARs) play essential roles in memory formation, neuronal plasticity and brain development with their dysfunction linked to a range of disorders from ischemia to schizophrenia. Zinc and pH are physiological allosteric modulators of NMDARs with GluN2A containing receptors inhibited by nanomolar concentrations of divalent zinc and by excursions to low pH. Despite the widespread importance of zinc and proton modulation of NMDARs, the molecular mechanism by which these ions modulate receptor activity has proven elusive. Here, we use cryo-electron microscopy to elucidate the structure of the GluN1/GluN2A NMDAR in a large ensemble of conformations under a range of physiologically relevant zinc and proton concentrations. We show how zinc binding to the amino terminal domain elicits structural changes that are transduced though the ligand-binding domain and result in constriction of the ion channel gate.

biophysics↗

bioRxiv2018

Analysis of seven putative Na+/H+ antiporters of Arthrospira platensis NIES-39 using transcription profiling and In-silico studies: an indication towards alkaline pH acclimation

Na+/H+ antiporters mediated pH regulation is one of the known mechanism(s), which advocates a possible role of the antiporters in the alkaline pH tolerance of Arthrospira platensis NIES-39 too. Seven putative Na+/H+ antiporters have been reported in A. platensis. We have characterized these seven antiporters, where A1, Q2, L2 and L6 belong to the CPA1 family whereas C5, D5 and O6 belong to CPA2 family through various In-silico analysis. Conserved domain analysis of these seven putative antiporters indicate the presence of nine different kinds of domains. Out of these nine domains, six domains function as monovalent cation-proton antiporters and two as the universal stress protein (Usp) category. Transcription profile of these seven antiporters was also generated at three different pH and time frames which showed a significant difference in the m-RNA levels at pH 7, 9 and at 11 along with a temporal pattern of the expression profile. The Insilico and the real time PCR analysis, put together, suggest the active participation of these seven putative Na+/H+ antiporters in alkaline pH homeostasis of this cyanobacterial strain where CPA1 subfamily antiporters play a major role.

molecular biology↗

bioRxiv2018

Copy number variants in clinical WGS: deployment and interpretation for rare and undiagnosed disease

PurposeCurrent diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, whole genome sequencing (WGS) has the potential to detect all genomic mutation types on a single platform and workflow. Here we sought to evaluate copy number variant (CNV) calling as part of a clinically accredited WGS test.\n\nMethodsUsing a depth-based copy number caller we performed analytical validation of CNV calling on a reference panel of 17 samples, compared the sensitivity of WGS-based variants to those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis, annotation, filtering, visualization of WGS based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.\n\nResultsWe found that CNV calls from WGS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed. This pipeline also enabled identification of cases of uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of some CNVs enabled break-point level resolution of genomic rearrangements and phasing of de-novo CNVs.\n\nConclusionRobust identification of CNVs by WGS is possible within a clinical testing environment, and further developments will bring improvements in resolution of smaller and more complex CNVs.

genomics↗

bioRxiv2018

Rapid Whole Genome Sequencing Decreases Morbidity and Healthcare Cost of Hospitalized Infants

BACKGROUNDGenetic disorders are a leading cause of morbidity and mortality in infants. Rapid Whole Genome Sequencing (rWGS) can diagnose genetic disorders in time to change acute medical or surgical management (clinical utility) and improve outcomes in acutely ill infants.\n\nMETHODSRetrospective cohort study of acutely ill inpatient infants in a regional childrens hospital from July 2016-March 2017. Forty-two families received rWGS for etiologic diagnosis of genetic disorders. Probands received standard genetic testing as clinically indicated. Primary end-points were rate of diagnosis, clinical utility, and healthcare utilization. The latter was modelled in six infants by comparing actual utilization with matched historical controls and/or counterfactual utilization had rWGS been performed at different time points.\n\nFINDINGSThe diagnostic sensitivity was 43% (eighteen of 42 infants) for rWGS and 10% (four of 42 infants) for standard of care (P=.0005). The rate of clinical utility for rWGS (31%, thirteen of 42 infants) was significantly greater than for standard of care (2%, one of 42; P=.0015). Eleven (26%) infants with diagnostic rWGS avoided morbidity, one had 43% reduction in likelihood of mortality, and one started palliative care. In six of the eleven infants, the changes in management reduced inpatient cost by $800, 000 to $2,000,000.\n\nDISCUSSIONThese findings replicate a prior study of the clinical utility of rWGS in acutely ill inpatient infants, and demonstrate improved outcomes and net healthcare savings. rWGS merits consideration as a first tier test in this setting.

clinical trials↗

bioRxiv2017

An interlaboratory study of complex variant detection

Next-generation sequencing (NGS) is widely used and cost-effective. Depending on the specific methods, NGS can have limitations detecting certain technically challenging variant types even though they are both prevalent in patients and medically important. These types are underrepresented in validation studies, hindering the uniform assessment of test methodologies by laboratory directors and clinicians. Specimens containing such variants can be difficult to obtain; thus, we evaluated a novel solution to this problem in which a diverse set of technically challenging variants was synthesized and introduced into a known genomic background. This specimen was sequenced by 7 laboratories using 10 different NGS workflows. The specimen was compatible with all 10 workflows and presented biochemical and bioinformatic challenges similar to those of patient specimens. Only 10 of 22 challenging variants were correctly identified by all 10 workflows, and only 3 workflows detected all 22. Many, but not all, of the sensitivity limitations were bioinformatic in nature. We conclude that Synthetic controls can provide an efficient and informative mechanism to augment studies with technically challenging variants that are difficult to obtain otherwise. Data from such specimens can facilitate inter-laboratory methodologic comparisons and can help establish standards that improve communication between clinicians and laboratories.

genetics↗

bioRxiv2017

Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility

A key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It has been shown that vertebrate dyneins movement becomes strikingly enhanced upon interacting with a cofactor named dynactin and one of several cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for this increase in transport efficiency are not well understood, largely due to a lack of structural information. We used cryo-electron tomography to visualize the first 3-dimensional structure of the intact dynein-dynactin complex bound to microtubules. Our structure reveals that the dynactin-cargo-adapter complex recruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the dynein motor organization closely resembles that of axonemal dynein, suggesting that cytoplasmic dynein and axonemal dyneins may utilize similar mechanisms to coordinate multiple motors. We propose that grouping dyneins onto a single dynactin scaffold promotes collective force production as well as unidirectional processive motility. These findings provide a structural platform that facilitates a deeper biochemical and biophysical understanding of dynein regulation and cellular transport.

cell biology↗

bioRxiv2017

Structural analyses reveal that the ATG2A-WIPI4 complex functions as a membrane tether for autophagosome biogenesis

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

biochemistry↗