Biology subjects

Mitra, J.

Publications and source records attributed to Mitra, J..

15 recordsLinked to original sources

bioRxiv2026

YhdP forms an envelope-spanning phospholipid-binding bridge in complex with DedD

The outer membrane of Gram-negative bacteria is built from phospholipids synthesised at the inner membrane, but how they cross the periplasm remains unresolved. YhdP is the leading candidate for anterograde phospholipid transfer and is predicted to form an envelope-spanning bridge. Here we purify YhdP at endogenous levels and show by cryo-electron microscopy that it contains a continuous cavity, alongside an additional density that proteomics identifies as the cell wall-binding protein DedD. Liposome co-sedimentation shows that the C-terminal helices of YhdP bind membranes directly, and single-molecule tracking reveals that endogenous YhdP is as immobile as an outer membrane protein, indicating an envelope-spanning architecture in vivo. Native mass spectrometry demonstrates that the YhdP groove accommodates all major classes of E. coli phospholipid without headgroup preference. DedD engages YhdP through its disordered region and increases lipid density at the groove entrance, suggesting a potential role in lipid loading.

microbiology↗

bioRxiv2024

Selective Inhibition of Cytosolic PARylation via PARG99: A Targeted Approach for Mitigating FUS-associated Neurodegeneration

Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) are characterized by complex etiologies, often involving disruptions in functions of RNA/DNA binding proteins (RDBPs) such as FUS and TDP-43. The cytosolic mislocalization and aggregation of these proteins are linked to accumulation of unresolved stress granules (SGs), which exacerbate the disease progression. Poly-ADP-ribose polymerase (PARP)-mediated PARylation plays a critical role in this pathological cascade, making it a potential target for intervention. However, conventional PARP inhibitors are limited by their detrimental effects on DNA repair pathways, which are already compromised in ALS. To address this limitation, we investigated a strategy focused on targeting the cytosolic compartment by expressing the cytosol-specific, natural PAR- glycohydrolase (PARG) isoform, PARG99. Using ALS patient derived FUS mutant induced pluripotent cells (iPSCs) and differentiated neurons, we observed elevated levels of FUS in insoluble fractions in mutant cells compared to mutation-corrected isogenic lines. The insoluble FUS as well as TDP-43 levels increased further in sodium arsenite-treated or oxidatively stressed cells, correlating with accumulation of unresolved SGs. Notably, both PARG99 and PARP inhibitors reduced SG formation and insoluble FUS levels, however, PARG99 treated cells exhibited significantly lower DNA damage markers and improved viability under oxidative and arsenite stress. This study highlights the potential of PARG99 as a cytosol-specific intervention to mitigate FUS-associated toxicity while preserving critical nuclear DNA repair mechanisms, offering a promising strategy for addressing the underlying pathology of ALS and potentially other SG-associated neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Fructose-2,6-bisphosphate restores TDP-43 pathology-driven genome repair deficiency in motor neuron diseases

TAR DNA-binding protein 43 (TDP-43) proteinopathy plays a critical role in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We recently reported that TDP-43 plays an essential role in DNA double-strand break (DSB) repair via the non-homologous end-joining (NHEJ) pathway. Here, we provide evidence that the brain of patients with ALS exhibit persistent DNA damage in the transcribed regions of the genome. While investigating the mechanistic basis, we found that the activity of polynucleotide kinase 3-phosphatase (PNKP) was severely impaired in the nuclear extracts of patient brains and TDP-43-depleted cells. PNKP is a key player in DSB repair within the transcribed genome, where its 3-phosphate termini processing activity is crucial for preventing persistent DNA strand breaks and neuronal death. The inactivation of PNKP was due to the reduced level of its interacting partner, phosphofructo-2-kinase fructose 2,6 bisphosphatase (PFKFB3), and its biosynthetic product, fructose-2,6-bisphosphate (F2,6BP), an allosteric modulator of glycolysis. Recently, we have demonstrated that F2,6BP acts as a positive modulator of PNKP activity in vivo. Furthermore, F2,6BP supplementation in cultured ALS patient-derived neural progenitor stem cells (NPSCs) reduced the toxic aggregation of polyubiquitinated TDP-43 and cytosolic pTDP-43 (S409/410). Notably, F2,6BP supplementation restored the PNKP activity in the nuclear extracts from autopsied ALS/FTD brain tissues and patient iPSC-derived NPSCs harboring TDP-43 mutations. Importantly, F2,6BP administration significantly restored the genome integrity and motor phenotypes in a Drosophila model of ALS-TDP-43. Collectively, these findings underscore the therapeutic potential of F2,6BP in TDP-43 pathology-associated motor neuron diseases.

neuroscience↗

bioRxiv2024

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

biochemistry↗

bioRxiv2023

Single molecule studies of the native hair cell mechanosensory transduction complex

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex. Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

biochemistry↗

bioRxiv2023

A comparative study of low-pH tolerance and chitinase activity between toxigenic and non-toxigenic strains of Vibrio cholerae

Cholera toxin, encoded by the ctx gene, is a key virulence factor in toxigenic Vibrio cholerae (ctx+) strains. However, some non-toxigenic V. cholerae (ctx-) strains are also pathogenic to humans and the mechanism involved in low-pH tolerance and pathogenicity in these strains remains unclear. To address this, we profiled the growth and chitinase activity in different pH of two clinical isolates of V. cholerae: VC20, a ctx+ strain, and WO5, a ctx- strain. We also compared the expression level of key genes involved in pathogenesis between the strains. WO5, the non-toxigenic strain had robust growth and greater chitinase activity across a wide pH range, in comparison to VC20. Additionally, WO5 expressed higher levels of transcripts from genes implicated in host cell adhesion and virulence, namely ompK and toxT, respectively. Notably, we propose that lower hapR levels in WO5 contrary to VC20 is key to its low-pH tolerance. To systematically identify genes involved in low pH tolerance, we used a sequence-based homology search and found a widespread presence of low-pH adaptation modules, lysine-cadaverine, and ornithine-putrescine in multiple representative species of the Vibrio phylum. Furthermore, our analysis suggests that the loss of a gene encoding nitrite reductase that confers low pH tolerance is specific to V. cholerae and V. mimicus. Together, these findings reveal that the low-pH tolerance enhances the chitinase activity of the non-toxigenic strain that could help V. cholerae to survive the acidic environment of the stomach and readily colonize the intestine.

microbiology↗

bioRxiv2022

Enhanced mTORC1 signaling and Protein Synthesis in Parkinson Disease Pathogenesis

Pathologic -syn destabilizes the TSC 1 and 2 complex leading to mTORC1 activation, enhanced protein translation and neurodegeneration in PD. AbstractPathological -synuclein (-syn) plays an important role in the pathogenesis of -synucleinopathies such as Parkinsons disease (PD). Disruption of protein homeostasis is thought be central to PD pathogenesis, however the molecular mechanism of this deregulation is poorly understood. Here we report that pathologic -syn binds to tuberous sclerosis protein (TSC) 2 and destabilizes the TSC1-TSC2 complex leading to activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and enhanced mRNA translation. Dopamine neuron loss, behavioral deficits and aberrant biochemical signaling in the -syn preformed fibril (PFF) and Drosophila -syn transgenic models of pathologic -syn induced degeneration were attenuated by genetic and pharmacologic inhibition of mTOR and protein translation. Our findings establish a potential molecular mechanism by which pathologic -syn activates mTORC1 leading to enhanced protein translation and concomitant neurodegeneration in PD.

neuroscience↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

The repeat region of the circumsporozoite protein is an elastic linear spring with a functional role in Plasmodium sporozoite motility

The circumsporozoite protein (CSP) forms a dense coat on the surface of the sporozoite, the infective stage of the malaria parasite. The central repeat region of CSP is a critical component of the only licensed malaria vaccine yet little is known about its structure or function. We found that sporozoite mutants with severely truncated or scrambled repeats have impaired motility due to altered adhesion site formation and dynamics, suggesting that the CSP repeats provide a cohesive environment in which adhesion sites can form. We hypothesized that biophysical properties of the repeats are important in this role and interrogated this using single-molecule fluorescence-force spectroscopy. We show that the repeats are a stiff, linear spring with elastic properties, dependent upon length and lost when the repeats are scrambled. These data are the first evidence that the CSP repeat region serves a functional role during infection and motility, likely mediated through its biophysical properties. SummaryNo clear function of the central repeat region of the malaria circumsporozoite protein has been described to date, despite its central role in the only licensed malaria vaccine. Here we use mutational analysis and single-molecule fluorescence-force spectroscopy to describe the structural properties and determine the function of this conserved region and important vaccine target. HighlightsThe CSP repeats have properties of a linear spring Scrambling or large truncations of the repeats leads to defects in sporozoite motility Motility defects are attributed to abnormal formation of adhesion sites

microbiology↗

bioRxiv2021

Orc6 at replication fork enables efficient mismatch repair

In eukaryotes, the Origin Recognition Complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for pre-replication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression post pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S-phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutS and enhances the chromatin-association of MutL, thus enabling efficient mismatch repair. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S-phase. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/443400v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@bfdaa8org.highwire.dtl.DTLVardef@1ac791dorg.highwire.dtl.DTLVardef@436d1corg.highwire.dtl.DTLVardef@b07689_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHuman Orc6 is dispensable for G1 licensing, but required for S-phase progression C_LIO_LIHuman Orc6 at the replication fork is an accessory factor for MMR complex C_LIO_LIDepletion of hOrc6 sensitizes cells to DNA damage and impairs ATR activation C_LIO_LIHuman Orc6 regulates MMR complex assembly and activity C_LI

molecular biology↗

bioRxiv2020

The genome of North American beaver provides insights into the mechanisms of its longevity and cancer resistance

The North American beaver (Castor canadensis) is an exceptionally long-lived and cancer-resistant rodent species, and thus an excellent model organism for comparative genomic studies of longevity. Here, we utilize a significantly improved beaver genome assembly to assess evolutionary changes in gene coding sequences, copy number, and expression. We found that the beaver Aldh1a1, a stem cell marker gene encoding an enzyme required for detoxification of ethanol and aldehydes, is expanded (~10 copies vs. two in mouse and one in human). We also show that the beaver cells are more resistant to ethanol, and beaver liver extracts show higher ability to metabolize aldehydes than the mouse samples. Furthermore, Hpgd, a tumor suppressor gene, is uniquely duplicated in the beaver among rodents. Our evolutionary analysis identified beaver genes under positive selection which are associated with tumor suppression and longevity. Genes involved in lipid metabolism show positive selection signals, changes in copy number and altered gene expression in beavers. Several genes involved in DNA repair showed a higher expression in beavers which is consistent with the trend observed in other long-lived mammals. In summary, we identified several genes that likely contribute to beaver longevity and cancer resistance, including increased ability to detoxify aldehydes, enhanced tumor suppression and DNA repair, and altered lipid metabolism.

genomics↗

bioRxiv2019

Real-time monitoring of cotranscriptional riboswitch folding and switching

Riboswitches function through cotranscriptional conformation switching governed by cognate ligand concentration, RNA folding and transcription elongation kinetics. To investigate how these parameters influence riboswitch folding, we developed a novel vectorial folding assay (VF) in which the superhelicase Rep-X sequentially liberates the RNA strand from a heteroduplex in a 5-to-3 direction, mimicking the nascent chain emergence during transcription. The RNA polymerase (RNAP)-free VF recapitulates the kinetically controlled cotranscriptional folding of a ZTP riboswitch, whose activation is favored by slower transcription, strategic pausing, or a weakened transcriptional terminator. New methods to observe positions and local rates of individual helicases show an average Rep-X unwinding rate similar to bacterial RNAP elongation (~60 nt/s). Real-time single-molecule monitoring captured folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These methods allow observation of individual folding RNAs as they occupy distinct folding channels within the landscape that controls gene expression and showed that riboswitch fate control is encoded in its sequence and is readily interpreted by a directionally moving protein even in the absence of an RNA polymerase.

biophysics↗

bioRxiv2019

Streamlining effects of extra telomeric repeat on telomere folding revealed by fluorescence-force spectroscopy

Tandem repeats of guanine rich sequences are ubiquitous in the eukaryotic genome. For example, in the human cells, telomeres at the chromosomal ends comprise of kilobases repeats of T2AG3. Four such repeats can form G-quadruplexes (GQs). Biophysical studies have shown that GQs formed from four consecutive repeats possess high diversity both in their structure and in their response to tension. In principle, a GQ can form from any four repeats that may not even be consecutive. In order to investigate the dynamics of GQ possessing such positional multiplicity, we studied five and six repeats human telomeric sequence using single molecule FRET as well as its combination with optical tweezers. Our results suggest preferential formation of GQs at the 3 end both in K+ and Na+ solutions although minority populations with a 5 GQ or long-loop GQs were also observed. Using a vectorial folding assay which mimics the directional nature of telomere extension, we found that the 3 preference holds even when folding is allowed to begin from the 5 side. Interestingly, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. Location of GQ on a long G-rich telomeric overhang and reduction in diversity of GQ conformations and mechanical responses through adjacent sequences have important implications in processes such as telomerase inhibition, alternative lengthening of telomeres, T-loop formation, telomere end protection and replication.

biophysics↗

bioRxiv2019

Nanomechanics and co-transcriptional folding of Spinach and Mango

Recent advances in fluorogen-binding RNA aptamers known as \"light-up\" aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn. Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K+, which is needed to stabilize the GQ cores, Mg2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.

biophysics↗

bioRxiv2018

Conformational heterogeneity allows access to DNA in longer Transcription Activator-Like Effector (TALE) arrays.

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. Here, we examine the kinetics of DNA binding for a set of TALE arrays with varying numbers of identical repeats using single molecule microscopy. Using a new deterministic modeling approach, we find evidence for conformational heterogeneity in both the free- and DNA-bound TALE arrays. Combined with previous work demonstrating populations of partly folded TALE states, our findings reveal a functional instability in TALE-DNA binding. For TALEs forming less than one superhelical turn around DNA, partly folded open states inhibit DNA binding. In contrast, for TALEs forming more than one turn, the partly folded open states facilitate DNA binding. Overall, we find that increasing repeat number results in significantly slower interconversion between the various DNA-free and DNA-bound states. These findings highlight the role of conformational heterogeneity and dynamics in facilitating macromolecular complex assembly.\n\nImpact StatementSingle molecule DNA-binding trajectories and deterministic modeling analyses demonstrate a functional role for high energy partly folded states in Transcription Activator-Like Effectors (TALEs) that could improve future TALEN design.

biophysics↗