bioRxiv Science⌕ Search

Biology subjects

Biswas, T.

Publications and source records attributed to Biswas, T..

At least 19 recordsLinked to original sources

A naturally evolved mutation in cavefish mc3r reshapes energy homeostasis across development in a nutrient-limited environment.

Food availability in natural habitats is often unpredictable and drives the selection of adaptive strategies in organisms to survive food scarcity. However, the genetic and molecular mechanisms that enable animals to survive under such nutritional extremes are poorly understood. Naturally adapted organisms therefore provide powerful genetic systems for uncovering the mechanisms that maintain energy homeostasis under nutritional extremes. Here, we leveraged the cavefish Astyanax mexicanus to identify a naturally occurring Thr166Met missense mutation in the melanocortin 3 receptor (mc3r) that reprograms energy homeostasis to facilitate adaptation to nutrient deprivation. To determine its functional consequences, we introduced the mutation into zebrafish mc3r using CRISPR/Cas9 genome editing. We show that adult mc3r mutants exhibit increased body size, adiposity, and hyperglycemia relative to wild-type animals, recapitulating key metabolic features of Tinaja cavefish and identifying mc3r as the first causal gene behind cavefish hyperglycemia. Investigations for early developmental consequences of the mutation revealed accelerated yolk utilization in mutant zebrafish larvae and strikingly elevated metabolic rates relative to WT, thus, uncovering a previously unrecognized role for mc3r in regulating energy expenditure. Importantly, the phenotype was also observed in Tinaja cavefish revealing a novel cavefish phenotype and establishing causal role for mc3r in elevated metabolic rate phenotype of cavefish. Together, these findings reveal that the Thr166Met variant produces distinct physiological consequences across life stages, enhanced energy storage and hyperglycemia in adulthood despite elevated larval energy expenditure. We propose that increased larval metabolism may support the energetic demands of enhanced foraging, whereas increased adiposity and blood glucose later in life provide energy reserves during periods of nutrient scarcity. More broadly, our study demonstrates how a single naturally evolved coding variant can reshape energy homeostasis across development and contribute to adaptation to nutrient-poor environments.

evolutionary biology↗

A Distal Super-Enhancer Regulate CD47 Expression and Promotes Macrophage-Mediated Phagocytosis of Ovarian Cancer Cells

CD47 is an innate immune checkpoint that can protect tumor cells from macrophage-mediated clearance, but the regulatory mechanisms underlying its expression in ovarian cancer (OC) remain incompletely understood. Here, we investigated the epigenetic regulation of CD47 expression in OC. Single-cell and spatial transcriptomic analyses indicated preferential CD47 expression in malignant epithelial cells. Integration of publicly available epigenomic datasets revealed a distal regulatory region with enhancer-associated features across ovarian cancer models and HGSOC patient samples. A genome-wide CRISPR-Cas9 screen further implicated PITX2 and EP300 as regulators of CD47 expression. Chromatin immunoprecipitation assays showed PITX2 and EP300 occupancy at candidate enhancer elements within this CD47 regulatory region. Comparative epigenomic analyses suggested that features of this regulatory landscape are also present in fallopian tube epithelial cells and become more prominent in OC. Together, these findings support a PITX2-EP300 regulatory axis associated with CD47 expression and provide evidence for its potential contribution to tumor immune evasion.

cancer biology↗

Cryo-EM reveals alternative modes of dimerization driving activation of IKK

The inhibitor of {kappa}B kinase (IKK) complex integrates diverse cellular inflammatory responses, and induces transcription factor NF-{kappa}B. The molecular mechanism by which IKK becomes catalytically active in response to signaling remains unclear despite structural knowledge of the individual IKK1/, IKK2/{beta}, and NEMO/IKK{gamma} protein components within its hetero-oligomeric assembly. Cryo-EM of the IKK2/{beta} homodimer bound to an associating NEMO/IKK{gamma} protein fragment, reveals multiple conformers. Mutual exclusivity of dimeric conformers, canonical versus alternate, is reflected in and dependent upon order-to-disorder transition of the canonical 6-helical bundle dimerization interface. Correlation of this unusual structural plasticity of IKK2/{beta} with its biochemical and cellular activities suggests mechanistic possibilities for how association with its partner scaffold protein NEMO/IKK{gamma} and polyubiquitin chains might dictate catalytic activation of IKK through distinct IKK2/{beta} conformers. SignificanceThe inhibitor of {kappa}B kinase (IKK) complex is central to inflammatory signaling via the NF-{kappa}B family transcription factors. Its activation mechanism has remained unclear. Cryo-EM analysis reveals that the constituent kinase IKK2/{beta} adopts structurally distinct, mutually exclusive dimeric conformations controlled by an ordered-to-disordered transition at its canonical dimerization interface. Stabilization of select IKK2/{beta} conformers by the scaffold protein NEMO in association with poly-ubiquitin chains is regulated through modular architecture and structural plasticity of distinctive kinase-associated domains, present only in kinases of this family. This unique regulatory mechanism governing catalytic activation of IKK2/{beta} provides a conceptual framework for targeting dysregulated NF-{kappa}B signaling in human diseases.

immunology↗

γLinolenic Acid Induces a Vitamin D Receptor-Independent Mineralization Program by Activating CaMKII--SMAD2/3 Pathway in Calvarial Osteoblasts

Vitamin D receptor (VDR) signaling is essential for osteoblast maturation and skeletal mineralization, yet the intracellular mechanisms linking VDR activity to matrix production remain poorly defined. Here, we show that vdr-/- calvarial osteoblasts initiate differentiation but fail to complete the transition to a mature, mineralizing state, exhibiting suppressed late-stage markers such as Dmp1, Phex, and Col1a1 and defective nodule formation both in vivo and in vitro. Transcriptomic profiling revealed a SMAD network imbalance, with elevated inhibitory SMADs and attenuated phosphorylation of both SMAD1/5/9 and SMAD2/3. Whereas SMAD1/5/9 activation was preserved in vivo and restored by exogenous BMP2 in vitro, consistent with in vivo paracrine BMP availability, reduced SMAD2/3 phosphorylation persisted in both contexts, indicating a cell-autonomous defect. Mechanistically, vdr-/- osteoblasts displayed impaired intracellular Ca2+ dynamics and diminished CaMKII activation, with VDR/RXR occupancy detected near the Camk2g locus. Pharmacological CaMKII inhibition (KN-93) recapitulated the phenotype, reducing SMAD2/3 phosphorylation and mineralization and establishing CaMKII as an upstream regulator of SMAD2/3. A milk-based diet rescued skeletal defects in vdr-/- mice in a calcium-independent manner and selectively restored CaMKII-SMAD2/3 signaling. Metabolomic profiling identified the omega-6 fatty acid y-linolenic acid (GLA) as an elevated circulating mediator; exogenous GLA restored Ca2+ flux, CaMKII activation, SMAD2/3 phosphorylation, matrix production, and mineralization, without reactivating Runx2, Sp7, or BMP-SMAD1/5/9 signaling, and dependent on CaMKII activity. These findings reveal a VDR-independent, GLA-inducible CaMKII-SMAD2/3 mineralization program, positioning metabolic modulation of calcium signaling as a strategy to restore osteoblast function under impaired vitamin D signaling.

physiology↗

LFCT: A Benchmark Dataset for Low-Frame-Rate Cell Tracking in Long-Term Live-Cell Microscopy

Cell tracking in time-lapse microscopy is essential for studying dynamic biological processes such as migration, proliferation, and lineage formation. Existing benchmarks primarily focus on high-framerate imaging, where short temporal intervals simplify correspondence between cells across consecutive frames. We present the Low Frame-rate Cell Tracking dataset (LCFT), a benchmark dataset designed specifically for evaluating cell tracking methods under low-frame-rate conditions. The dataset contains multi-day live-cell microscopy sequences from four human cell lines (MCF10A, MDA-MB-231, HEK293T, and U87), acquired at 10x and 20x magnifications using phase-contrast and fluorescence imaging (nucleus). Ground-truth annotations include cell identifications, temporal linking, lineage relationships, and mitosis events. To generate reliable annotations, automated segmentation and tracking were combined with extensive manual curation. LCFT provides a comprehensive resource for developing and benchmarking robust cell tracking algorithms capable of handling sparse temporal sampling and large inter-frame motion in long-term live-cell imaging experiments.

cell biology↗

A Toxic Tau-PFKFB3 Circuit Reduces F2,6BP Levels and Drives Neurodegeneration

Alzheimers disease (AD) and related dementias are progressive neurodegenerative disorders manifested by aggregation of Tau and Amyloid beta (A{beta}). Emerging evidence suggests that metabolic dysregulation contributes to AD pathogenesis, yet how metabolic alterations interface with neuronal integrity remains unclear. Here, we identify dysfunction in PFKFB3-F2,6BP (fructose-2,6-bisphosphate) metabolic axis as a key feature of AD. We show that pathological Tau aggregates aberrantly sequester PFKFB3, limiting its activity and resulting in F2,6BP depletion. F2,6BP exerts protective effects through multiple convergent mechanisms: (i) direct activation of polynucleotide kinase 3-phosphatase (PNKP) to facilitate DNA strand break repair; (ii) transcriptional upregulation of the protein phosphatase 2A catalytic subunit (PP2CA) to limit Tau phosphorylation; (iii) stabilization of PFKFB3 to diminish its sequestration into aggregates; and (iv) direct inhibition of Tau aggregation. These findings establish F2,6BP as a central node linking metabolic regulation to both genomic stability and proteostasis in AD. Importantly, exogenous F2,6BP supplementation rescues multiple pathological features across diverse model systems, including induced neuronal cell lines (iN), primary neurons, organotypic hippocampal slice cultures, and in a Drosophila model of AD. These findings redefine F2,6BP as a metabolite that directly coordinates genome maintenance and proteostasis in neurons. Overall, this study identifies the PFKFB3-F2,6BP axis as a central driver of AD pathogenesis and a promising therapeutic target. HighlightsO_LITau aggregates sequester PFKFB3 depletes neuronal F2,6BP C_LIO_LIF2,6BP links metabolism to DNA repair and Tau proteostasis C_LIO_LIF2,6BP activates PNKP and upregulates PP2A to counter Tau pathology C_LIO_LIF2,6BP supplementation rescues AD phenotypes across models C_LI

neuroscience↗

FeSBCP Analogue from Cyanobacteria: Insights from in vitro and in silico Studies

The blue light responsive Cryptochrome/Photolyase Family harbours two important photoactivatable proteins - cryptochromes and photolyases. While cryptochromes are essentially photoreceptors with diverse biological activities including circadian rhythm, photolyases repair UV damaged cyclobutane pyrimidine dimers (CPD) or (6-4) pyrimidine-pyrimidone photoadducts. Both cryptochromes and photolyases share a common Photolyase Homology Region (PHR) where they harbour a light harvesting antenna chromophore and redox-active flavin di-nucleotide (FAD). Even though photolyases do not possess an extension in the C-terminal region unlike cryptochromes, a recent discovery of FeS cluster containing bacterial cryptochrome-photolyase proteins (FeSBCPs) present an interesting retreat from the conventional photolyase architecture. They possess an additional modular redox center, [4Fe4S] iron-sulfur cluster connected by conserved cysteine residues. Herein, we characterize a cyanobacterial FeSBCP from Cyanobium sp. using tools from bioinformatics, biochemistry and biophysics. Sequence analysis reveals substitution in the well-conserved aromatic residues, meant for electron transfer. In silico modeling and docking supported by electrophoretic mobility shift assay as well as spectroscopic measurements do suggest efficient binding and repair of CyPhrB to damaged single/double-stranded substrates containing 6-4 photolesion. Considering FeS clusters established roles in DNA binding/repair activities, we speculate the role of FeS clusters in FeSBCPs and existence of FeS-FAD-DNA triangle towards efficient electron transfer.

biochemistry↗

Passive muscle forces in Drosophila are large but insufficient to support a flys weight

Movement of a limb is shaped by active forces generated by muscle contraction but also by passive forces within individual muscles and joints. In small animals such as insects, the contribution of passive forces to limb movement can match the active forces. However, most measurements of passive forces are limited to the femur-tibia joint in large insects. Here we take advantage of genetic tools in Drosophila to measure passive torques at multiple joints in the flys leg. We genetically inactivate all the motor neurons to assess passive forces. We find that the passive torques are well approximated by a linear spring, i.e., the passive torques linearly increase with angular deviation from the rest angle. The torques are much larger than the gravitational torque due to the leg itself. We estimate that the passive torques are seventy times smaller than necessary to support the weight of the animal. We also inactivated all the motor neurons in a freely standing fly and found that, as predicted from the model, the fly falls when the motor neurons are inactivated. We found that the height at which a fly stands, and, therefore the active forces vary. The flys height affects the time to initiate a fall. The time it takes for the fall is consistent with the active forces decaying with a time constant of [~]100 ms. Thus, although passive forces are strong and will have a large effect on limb kinematics, they are not strong enough to support the weight of the fly.

neuroscience↗

JNK2 pathway in colonocytes enhances gut barrier integrity in response to microbial acetate.

Weaning involves a nutritional shift from fat-rich milk to carbohydrate-based solid food, reshaping metabolism, microbiota, and gut immune tolerance. While dairy remains a component of the human diet beyond weaning, the impact of continued milk supplementation on gut epithelial homeostasis remains poorly understood. Here, using a mouse model, we show that continued milk-based feeding post-weaning promotes intestinal barrier function by enriching the commensal bacterium Dubosiella newyorkensis, which produces acetate to activate epithelial JNK2 signaling. This pathway enhances barrier integrity and suppresses inflammation induced by mild Dextran Sodium Sulphate (DSS) treatment. In contrast, feeding a lard-based high-fat diet or transient pharmacologic inhibition of JNK2 induces epithelial P38 activation, resulting in barrier disruption and inflammation. Importantly, the beneficial effects of milk were observed only if initiated during the weaning period, when the microbiome is in a metastable transitional state. Initiation of the same intervention two weeks after weaning led to P38 activation and inflammatory responses. These findings highlight the microbiome-dependent, stage-specific effects of diet and underscore the importance of early-life nutritional interventions for long-term gut health.

physiology↗

From the fly connectome to exact ring attractor dynamics

A cognitive compass enabling spatial navigation requires the neural representation of head direction (HD), yet the neural circuit architecture enabling this representation remains unclear. While various network models have been proposed to explain HD systems, these models rely on simplified circuit architectures that may be irreconcilable with empirical observations from connectomes. Here we construct a neural network model for the fruit fly HD system that satisfies both connectome-derived architectural constraints and the functional requirement of continuous head-direction representation. To achieve this, we first characterized an ensemble of continuous attractor networks where compass neurons providing local mutual excitation are coupled to inhibitory neurons. Our multipopulation model allowed us to discover a new class of ring attractor network with weaker symmetry requirements than usually assumed. For each of the four available fly connectomes, our analyses uncovered three distinct realizations of these networks. Furthermore, we found that synaptic variations among and around the fly connectomes can be compensated by cell-type-specific rescaling of synaptic weights, which could be potentially achieved through neuromodulation. The fly connectomes are ideally configured to take advantage of this mechanism, suggesting a novel design principle linking synapse-resolution connectivity to network computation.

neuroscience↗

Genetically-Encoded Fluorescence Barcodes for Single-Cell Analysis

Genetically-encoded, single-cell barcodes are broadly useful for experimental tasks such as lineage tracing or genetic screens. For such applications, a barcode library would ideally have high diversity (many unique barcodes), non-destructive identification (repeated measurements in the same cells or population), and fast, inexpensive readout (many cells and conditions). Current nucleic acid barcoding methods generate high diversity but require destructive and slow/expensive readout, and current fluorescence barcoding methods are non-destructive, fast, and inexpensive to readout but lack high diversity. We recently proposed theory for how fluorescent protein combinations may generate a high-diversity barcode library with non-destructive, fast and inexpensive identification. Here, we present an initial experimental proof-of-concept by generating a library of [~]150 barcodes from two-way combinations of 18 fluorescent proteins, 61 of which are tested experimentally. We use a pooled cloning strategy to generate a barcode library that is validated to contain every possible combination of the 18 fluorescent proteins. Experimental results using single mammalian cells and spectral flow cytometry demonstrate excellent classification performance of individual fluorescent proteins, with the exception of mTFP1, and of most evaluated barcodes, with many true positive rates >99%. The library is compatible with genetic screening for hundreds of genes (or gene pairs) and lineage tracing hundreds of clones. This work lays a foundation for greater diversity libraries (potentially [~]105 and more) generated from hundreds of spectrally-resolvable tandem fluorescent protein probes.

synthetic biology↗

3D genome organization under extreme metabolic adaptation shows variations in genome compartmentalization and looping

The cave morphs of Astyanax mexicanus have evolved a suite of distinct adaptations to life in perpetual darkness, including the loss of eyes and pigmentation loss, as well as profound metabolic changes such as hyperphagia and starvation resilience, traits that sharply contrast with those of their river-dwelling surface counterparts. While changed gene expression is a primary driver of these adaptations, the underlying role of 3D genome organization - a key regulator of gene expression - remains unexplored. Here, we investigate the 3D genome architecture of the livers of surface fish and two cavefish morphs (Pachon and Tinaja) using Hi-C, performing the first comparative 3D genomic analysis in this species. We analyzed and identified cave-specific 3D genomic features, such as genomic compartments and loops, which were conserved in both the cave populations but absent in surface fish. Integrating the 3D genome data with transcriptomic and epigenetic datasets, linked these changes to differential expression of metabolically relevant genes, such as Arhgef19 and Endog. Additionally, our study also uncovered genomic inversions unique to cavefish, potentially tied to cave adaptation. Our findings suggest that 3D genome organization contributes to transcriptomic shifts underlying cavefish phenotypes, providing a novel intra-species and morph specific perspective on 3D chromatin evolution. This study establishes a foundation for exploring how genome architecture potentially facilitates adaptation to new environments. Comparison of morphs within the same species also establishes a foundation for better understanding of how 3D genome reorganization may drive speciation and phenotypic diversity.

evolutionary biology↗

Leg compliance is required to explain the ground reaction force patterns and speed ranges in different gaits

Two simple models - vaulting over stiff legs and rebounding over compliant legs - are employed to describe the mechanics of legged locomotion. It is agreed that compliant legs are necessary for describing running and that legs are compliant while walking. Despite this agreement, stiff legs continue to be employed to model walking under the assumption that the compliance of the leg during walking is high enough to be considered stiff. Here we study gait choice and walk-to-run transition in a biped with compliance and show that the principles underlying gait choice and transition are completely different from stiff legs. Two findings underpin our conclusions: First, at the same speed, step length, and stance duration, multiple gaits that differ in the number of leg contraction cycles are possible. Among them, humans and other animals choose the (normal) gait with M-shaped vertical ground reaction forces (vGRF) not just because of energy considerations but also constraints from forces. Second, the transition from walking to running occurs because of three factors: vGRF minimum at mid-stance characteristic of normal walking, synchronization of horizontal and vertical motions during single support, and velocity redirection during the double support. The insight above required an analytical approximation of the double spring-loaded pendulum (DSLIP) model describing the intricate oscillatory dynamics that relate single and double support phases. Additionally, we also examined DSLIP as a quantitative model for locomotion and conclude that DSLIP speed-range is limited. However, insights gleaned from the analytical treatment of DSLIP are general and will inform the construction of more accurate models of walking.

neuroscience↗

Cooperativity among clustered κB sites within promoters and enhancers dictates transcriptional specificity of NF-κB RelA along with specific cofactors

Non-consensus binding sites of transcription factors are often observed within the regulatory elements of genes; however, their effect on transcriptional strength is unclear. Within the promoters and enhancers of NF-{kappa}B-responsive genes, we identified clusters of non-consensus {kappa}B DNA sites, many exhibiting low affinity for NF-{kappa}B in vitro. Deletion of these sites demonstrated their collective critical role in transcription. We explored how these "weak" {kappa}B sites exert their influence, especially given the typically low nuclear concentrations of NF-{kappa}B. Using proteomics approaches, we identified additional nuclear factors, including other DNA-binding TFs, that could interact with {kappa}B site-bound NF-{kappa}B RelA. ChIP-seq and RNA-seq analyses suggest that these accessory TFs, referred to as the TF-cofactors of NF-{kappa}B, facilitate dynamic recruitment of NF-{kappa}B to the clustered weak {kappa}B sites. Overall, the occupancy of NF-{kappa}B at promoters and enhancers appears to be defined by a collective contribution from all {kappa}B sites, both weak and strong, in association with specific cofactors. This congregation of multiple factors within dynamic transcriptional complexes is likely a common feature of transcriptional programs. SIGNIFICANCEThe NF-{kappa}B RelA dimers undergo rapid activation by cytokines and pathogens, driving expeditious expression of target genes upon binding to DNA elements known as {kappa}B sites, located in the regulatory regions. We find that promoter and enhancer regions of RelA target genes harbor multiple {kappa}B sites, most being non-consensus with minimal affinity to NF-{kappa}B in vitro. Recruitment of RelA dimer in vivo depend on these {kappa}B sites, weak and strong, and appears to be regulated by various accessory factors, including other DNA-binding transcription factors. Overall, this study points to a coordinated network of factors communicating with both weak and strong {kappa}B sites to recruit RelA dimers, enabling rapid gene activation.

biochemistry↗

Phosphorylation-induced flexibility of proto-oncogenic Bcl3 regulates transcriptional activation by NF-κB p52 homodimer

B cell lymphoma 3 (Bcl3), a member of the I{kappa}B family proteins, modulates transcription by primarily associating with NF-{kappa}B p50 and p52 homodimers. Bcl3 undergoes extensive phosphorylation, though the functions of many of these modifications remain unclear. We previously described that phosphorylation at Ser33, Ser114 and Ser446 partially switches Bcl3 from acting as an I{kappa}B-like inhibitor to a transcription regulator by associating with the (p52:p52):DNA binary complex. Here, we identified another critical phosphorylation site, Ser366. Substituting at all four residues to phospho-mimetic glutamate further enhances Bcl3s transcriptional activity. Phospho-modifications retain Bcl3s ability to stably bind p52 but induces reciprocal structural changes as revealed by HDX-MS experiments; the N-terminal region stiffens, while the C-terminus becomes more flexible. The increased flexibility allowed the Bcl3:(p52p52) binary complex to better accommodate DNA. The removal of the C-terminal 28-residues transformed Bcl3 into a transcriptional activator independent of phosphorylation. Notably, most identified mutations in Bcl3 from various cancers map to its C-terminus, suggesting the functional relevance of Bcl3 C-terminal structural flexibility and enhanced interaction with (p52p52):DNA complex to transcriptional potential and disease. Overall, this study uncovers the mechanistic basis by which phosphorylation-driven structural changes convert Bcl3 from an inhibitor to a transcriptional cofactor of NF-{kappa}B, and how deregulation of its activity through altered phosphorylation or mutation can lead to cancer.

biochemistry↗

Potential role of microRNAs in regulating transcriptional profile, and sculpting development and metabolism in cavefish.

Astyanax mexicanus, a species with both surface-dwelling and multiple cave-dwelling populations, offers a unique opportunity to study repeated adaptation to dark and resource-scarce environments. While previous work has identified large-scale changes in gene expression between morphs even under identical laboratory conditions, the regulatory basis of these expression differences remains largely unexplored. In this study, we focus on microRNAs (miRNAs) as key regulators of gene expression to understand cavefish adaptation nuances. Our analysis identified 683 miRNAs, which not only surpasses the number documented in related species but also provides the first comprehensive catalog of miRNAs for this species. We identified a unique subset of differentially expressed miRNAs common to all studied cave-dwelling populations, potentially orchestrating the nuanced gene expression patterns required for survival in the challenging cave milieu. Gene Ontology analysis of the predicted miRNA targets revealed involvement in developmental and metabolic pathways that are pivotal for thriving in nutrient-limited environments, such as the regulation of neuromast migration. Moreover, our study provides evidence for miRNA influence on circadian rhythm and oxidative stress response, both essential adaptations for the cave-dwelling lifestyle. The comprehensive miRNA catalog generated will guide future investigations into the intricate world of miRNA-mediated evolution of complex traits.

evolutionary biology↗

A glycolytic metabolite restores DNA repair activity of polynucleotide kinase 3-phosphatase in polyglutamine (PolyQ) diseases

Huntingtons disease (HD) and spinocerebellar ataxia type 3 (SCA3) are the two most prevalent polyglutamine (polyQ) neurodegenerative diseases, caused by CAG (encoding glutamine) repeat expansion in the coding region of the huntingtin (HTT) and ataxin-3 (ATXN3) proteins, respectively. We have earlier reported that the activity, but not the protein level, of an essential DNA repair enzyme, polynucleotide kinase 3-phosphatase (PNKP), is severely abrogated in both HD and SCA3 resulting in accumulation of double-strand breaks in patients brain genome. While investigating the mechanistic basis for the loss of PNKP activity and accumulation of DNA double-strand breaks leading to neuronal death, we observed that PNKP interacts with the nuclear isoform of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Depletion of PFKFB3 markedly abrogates PNKP activity without changing its protein level. Notably, the levels of both PFKFB3 and its product fructose-2,6 bisphosphate (F2,6BP), an allosteric modulator of glycolysis, are significantly lower in the nuclear extracts of post-mortem brain tissues of HD and SCA3 patients. Supplementation of F2,6BP restored PNKP activity in the nuclear extracts of patients brain. Moreover, intracellular delivery of F2,6BP restored both the activity of PNKP and the integrity of transcribed genome in neuronal cells derived from striatum of HD mouse. Importantly, supplementing F2,6BP rescued the HD phenotype in Drosophila, suggesting F2,6BP to serve in vivo as a cofactor for the proper functionality of PNKP and thereby, of brain health. Our results thus provide a compelling rationale for exploring the therapeutic use of F2,6BP and structurally related compounds for treating polyQ diseases. SignificanceTo unravel the biological basis for the loss of PNKP activity in HD and SCA3, the two most prevalent polyglutamine neurodegenerative disorders, we analyzed PNKP interactome and found that the nuclear isoform of a glycolytic enzyme PFKFB3 associated with PNKP and other repair proteins forming a multiprotein complex. Surprisingly, we found that PFKFB3 and its biosynthetic product, F2,6BP are significantly low in the affected region of patients brain. Exogenous addition of F2,6BP restored PNKP activity in patients brain nuclear extract. Moreover, supplementing F2,6BP in HD cells and fruit flies restored genome integrity and rescued the disease symptoms. While there is no curative therapy for HD/SCA3, except symptom management, our discovery suggests that F2,6BP supplementation would be a promising therapeutic option.

biochemistry↗

Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement

Diversity-generating retroelements (DGRs) create massive protein sequence variation in ecologically diverse microbes. Variation occurs during reverse transcription of a protein-encoding RNA template coupled to misincorporation at adenosines. In the prototypical Bordetella bacteriophage DGR, the template must be surrounded by upstream and downstream RNA segments for cDNA synthesis by the reverse transcriptase bRT and associated protein Avd. The function of the surrounding RNA was unknown. Cryo-EM revealed that this RNA enveloped bRT and lay over barrel-shaped Avd, forming an intimate ribonucleoprotein (RNP). An abundance of essential interactions between RNA structural elements and bRT-Avd precisely positioned an RNA homoduplex for initiation of cDNA synthesis by cis-priming. Our results explain how the surrounding RNA primes cDNA synthesis, promotes processivity, terminates polymerization, and strictly limits mutagenesis to select proteins through mechanisms that are likely conserved in DGRs from distant taxa.

microbiology↗