Biology subjects

Ghosh, G.

Publications and source records attributed to Ghosh, G..

At least 19 recordsLinked to original sources

bioRxiv2026

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↗

bioRxiv2026

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↗

bioRxiv2025

Modeling collective cell migration in a heterogeneous environment: Drosophila border cell migration as a model paradigm

Collective cell migration underpins development and disease progression, yet the influence of heterogeneous microenvironments on intercellular forces and migration dynamics remains elusive. Focusing on the chemotactic migration of border cell clusters in Drosophila egg chambers, we present a computational framework that integrates intercellular force dynamics and cell shape changes during large-scale migration, validated with live imaging data. Our model replicates in vivo observations, such as rotations of the cell cluster and its tendency to follow a central path in the egg chamber. Using the model, we discover instantaneous acceleration/deceleration phases as the cell cluster migrates through junctions of other cell types. We also predict how environmental tissue topology and perturbations in chemoattractant gradients and adhesion impact cluster speed and rotation. By addressing the limitations of prior models of border cell migration, our approach offers broad applicability to other migration contexts involving interactions among different cell types in complex environments.

biophysics↗

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

F2,6BP restores mitochondrial genome integrity in Huntingtons Disease

Several reports have indicated that impaired mitochondrial function contributes to the development and progression of Huntingtons disease (HD). Mitochondrial genome damage, particularly DNA strand breaks, is a potential cause for its compromised functionality. Here we show that the activity of polynucleotide kinase 3-phosphatase (PNKP), a critical DNA end-processing enzyme, is significantly decreased in the mitochondrial extract of HD patients brains due to a lower level of fructose-2,6 bisphosphate (F2,6BP), a biosynthetic product of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Such decrease in PNKP activity leads to persistent DNA strand breaks that are refractory to subsequent steps for repair completion. Both PFKFB3 and F2,6BP, an allosteric modulator of glycolysis, are also present in the mitochondria and PFKFB3 is part of a mitochondrial DNA repair complex containing HTT, PNKP, DNA Pol {gamma} (POLG) and Lig III. Notably, PNKP binds F2,6BP (Kd= 525{+/-}25 nM) and utilizes it as a cofactor. The levels of both F2,6BP and PFKFB3 are significantly decreased in the mitochondrial extract of HD mouse striatal neuronal cells and patients brain. Activity of PNKP is thus severely decreased in the mitochondrial extract; however, addition of F2,6BP restored its activity. Moreover, supplementation of F2,6BP in HD cells restored PFKFB3 level, mitochondrial genome integrity and partially restored mitochondrial membrane potential, mitochondrial respiration and prevented pathogenic aggregate formation. We also observed that supplementation with F2,6BP restored mitochondrial genome integrity in an HD Drosophila model. Our findings, therefore, suggest that F2,6BP-mediated restoration of PNKP activity could have a profound impact in ameliorating neurodegenerative symptoms in HD. SignificanceWe reported earlier the loss of PNKP activity in the nuclear extracts from HD patients brain. However, a glycolytic metabolite, F2,6BP, can restore PNKP activity and rescue organismal phenotypes in HD fly models. As PNKP is present in mitochondria and several reports indicate that mitochondrial dysfunction contributes to HD, we therefore analyzed PNKP activity in the mitochondrial extract. Surprisingly, we found that PFKFB3 and its product, F2,6BP are present in mitochondria, but significantly low in patients brains. Exogenous addition of F2,6BP restored PNKP activity in patients brain mitochondrial extract. Moreover, supplementing F2,6BP in HD cells and fruit flies restored mitochondrial genome integrity suggesting maintaining adequate intracellular F2,6BP levels is critical for proper functionality of PNKP and thereby of brain health.

neuroscience↗

bioRxiv2024

Tumor suppressor NME1/NM23-H1 modulates DNA binding of NF-κB RelA

The dimeric NF-{kappa}B family of transcription factors activates transcription by binding sequence-specifically to DNA response elements known as {kappa}B sites, located within the promoters and enhancers of their target genes. While most NF-{kappa}B remain inactive in the cytoplasm of unstimulated cells, a small amount of RelA, one of its members, persists in the nucleus, ensuring low-level expression of genes essential for homeostasis. Several cofactors have been identified that aid in DNA binding of RelA. In this study, we identify NME1 (nucleoside diphosphate kinase 1) as a cofactor that enhances RelAs ability to bind {kappa}B sites within the promoters of a subset of its target genes, promoting their expression under both unstimulated and stimulated conditions. Depletion of NME1 influences activation or repression of several genes that are unresponsive to TNF, despite containing {kappa}B sites in their promoters but not in clusters. This suggests that clustering of kB sites may be necessary for RelA-dependent transcription complex assembly. NME1 appears to act as a cofactor for other transcription factors to regulate these genes. NME1 does not directly contact {kappa}B DNA but interacts with RelA, with this interaction being further strengthened in the presence of {kappa}B DNA. Notably, NME1 alone has a marginal effect in enhancing RelAs DNA binding, suggesting that NME1 likely cooperate with other cofactors to regulate DNA binding and transcription through RelA. These observations underscore the intricate assembly of transcription complexes centered on NF-{kappa}B.

biochemistry↗

bioRxiv2024

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↗

bioRxiv2024

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↗

bioRxiv2024

Identification of Candidate Protein Biomarkers Associated with Domoic Acid Toxicosis in Cerebrospinal Fluid of California Sea Lions (Zalophus californianus)

Since 1998, California sea lion (Zalophus californianus) stranding events associated with domoic acid toxicosis have consistently increased. Outside of direct measurement of DA in bodily fluids at the time of stranding, currently there are no practical non-lethal clinical tests for the diagnosis of domoic acid toxicosis (DAT) that can be utilized in a large-scale rehabilitation facility. Proteomic analysis was conducted to discover candidate protein markers of DAT using cerebrospinal fluid from stranded California sea lions with acute DAT (n = 8), chronic DAT (n = 19), or without DAT (n = 13). A total of 2005 protein families were identified experiment-wide (FDR < 0.01). Of these proteins, 83 were significantly different in abundance across the three groups (adj. p < 0.05). Cytoplasmic malate dehydrogenase (MDH1), 5-3 exonuclease PLD3, disintegrin and metalloproteinase domain-containing protein 22 (ADAM22), 14-3-3 protein gamma (YWHAG), neurosecretory protein VGF, and calsyntenin-1 (CLSTN1) were able to discriminate California sea lions with or without DAT (ROC > 0.75). Immunoglobulin kappa light chain-like (IGKV2D-28), receptor-type tyrosine-phosphatase F (PTRPF), kininogen-1 (KNG1), prothrombin (F2), and beta-synuclein (SNCB) were able to discriminate acute DAT from chronic DAT (ROC > 0.75). Interestingly, proteins involved in alpha synuclein deposition were over- represented as classifiers of DAT and many of these proteins have been implicated in a variety of neurodegenerative diseases. These proteins should be considered potential markers for DAT in California sea lions, as well as markers to discriminate between acute or chronic DAT, and should be considered priority for future validation studies as biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD041356 (http://proteomecentral.proteomexchange.org/dataset/PXD041356).

biochemistry↗

bioRxiv2023

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↗

bioRxiv2023

Ecdysone Orchestrates Notch and Broad Symphony to Craft Epithelial Cell Shape Change

Morphogenesis in the metazoans relies on cell shape transformations that forms an integral component of organ development, form generation and maintenance of tissue homeostasis. Nevertheless, a comprehensive grasp of how the epithelial morphogenesis is modulated in the metazoans remain still elusive. The Steroid hormones play a pivotal role in morphogenesis spanning several organs including the gonads, urogenital tracts, and mammary glands. Employing the Drosophila oogenesis model, we investigated the the role of steroid hormone receptor, Ecdysone receptor (EcR) involvement in transforming anterior epithelial follicle cells (AFCs) from cuboidal to squamous shape. Consistent with the fact that the activity of EcR in the AFCs coincides with the timing of cuboidal-to-squamous shape transition, we found that depletion of EcR function impedes the shape transformation of AFCs. We report that EcR doesnt impair the follicle cell fate, but impedes the morphological change by restricting the remodelling of lateral and adherens junctions. Employing the classical genetic tools and immnohistochemistry, we show that EcR limits the Notch-Broad axis to facilitate alteration of the shape of AFCs. Our study suggests a mechanistic model where Ecdysone signalling, via the Notch pathway, finetunes the activity of non-muscle myosin heavy chain zipper, prompting AFC shape transition. In sum, our work illuminates how Ecdysone signalling orchestrates epithelial follicle cell morphogenesis during metazoan development.

developmental biology↗

bioRxiv2023

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IkappaBalpha without requiring ATP

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of I{kappa}B by IKK2, a prototypical Ser/Thr kinase, is critical for canonical NF-{kappa}B activation. Here, we report that IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG+1 (DLG in IKK1) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its I{kappa}B substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of I{kappa}B at S32 or S36, but rather directs phosphorylation of I{kappa}B at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to I{kappa}B in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop, and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-{kappa}B activation.

biochemistry↗

bioRxiv2022

TOR signalling regulates epithelial cell shape transition in Drosophila oogenesis

Epithelial morphogenesis plays an important role in form generation, organ development, and maintenance of adult tissues in the metazoans. Given its wide implication, aberrant morphogenesis is linked to severe developmental defects and in a few instances also associated with tumorigenesis. Employing the model of Drosophila oogenesis, we have examined the role of the evolutionary conserved Target of Rapamycin (TOR) kinase pathway, a known regulator of cell growth and size in mediating shape transition of cuboidal cells to squamous epithelial fate. Utilizing genetic tools, immunohistochemistry, and live cell imaging, we demonstrate that TOR signaling is active and required for epithelial morphogenesis during Drosophila oogenesis. Further, loss of function analyses indicates that non-canonical TOR signaling functions through PAR-1 to mediate the removal of lateral cell adhesion molecule, Fasciclin 2, to allow proper squamous cell morphogenesis. In addition, we demonstrate the effect of TOR through PAR-1 on cell shape transition is mediated via the modulation of endocytosis. Overall, our data give novel insight into how TOR signaling mediates cell shape transition during epithelial morphogenesis in the metazoans.

developmental biology↗

bioRxiv2022

Germline protein, Cup, non-cell autonomously limits migratory cell fate in Drosophila oogenesis.

Attaining migratory fate from a stationary cell population is complex and indispensable both for the multicellular organism development as well for the pathological condition like tumor metastasis. Though widely prevalent in the metazoans, the molecular understanding of this phenomenon remains elusive. Specification of migratory border cells from the follicular epithelium during Drosophila oogenesis has emerged as one of the excellent model systems to study how motile cell are specified. JAK-STAT activation in 6-10 anterior most follicle cells of the Drosophila egg chamber transforms them to a migratory cluster called the border cells. We show that a nurse cell protein, Cup, non-cell autonomously restricts the domain of JAK-STAT activation in the anterior follicle cells. Further examination suggests that Cup functions through Rab11GTPase to regulate Delta trafficking in the nurse cells potentiating Notch activation in the anterior follicle cells. Since Notch activity in the follicle cells modulates the JAK-STAT, any perturbation in Notch activation affects the border cell fate. Altogether, we propose that germline Cup affects the border cell fate through appropriate activation of Notch and JAK-STAT signaling in the follicle cells.

developmental biology↗

bioRxiv2022

Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation

The mammalian NF-{kappa}B p52:p52 homodimer together with its cofactor Bcl3 activates transcription of {kappa}B sites with a central G/C base pair (bp), while it is inactive toward {kappa}B sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined its structure in complex with a P-selectin(PSel)-{kappa}B DNA (5-GGGGTGACCCC-3) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-{kappa}B-DNA complex structures, which have a central A/T bp. Molecular dynamics (MD) simulations show free DNAs exist in distinct preferred conformations, and p52:p52 homodimer induces the least amount of conformational changes on the more transcriptionally active natural PSel-{kappa}B DNA in the bound form. Our binding assays further demonstrate that the fast kinetics driven by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for {kappa}B DNA in complex with NF-{kappa}B and suggest the importance of binding kinetics, dictated by free DNA conformational and dynamic states, in controlling transcriptional activation for NF-{kappa}B.

biochemistry↗

bioRxiv2021

Mechanisms of modulation of pre-mRNA 3D structural scaffolds for splicing substrate definition

We recently reported that serine-arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65, and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

biochemistry↗

bioRxiv2021

Chromogranin A plays a crucial role in the age-related development of insulin resistance and hypertension

Aging is associated with the development of metabolic disorders, including insulin resistance and hypertension. Young mice that are negative for the neuroendocrine prohormone Chromogranin A (CgA knockout, CgA-KO) display two opposite aging phenotypes: hypertension but heightened insulin sensitivity. We determined these phenotypes in aging CgA mice. In comparison, aging wild-type (WT) mice gradually lost glucose tolerance and insulin sensitivity. Moreover, while aging WT mice had increased inflammation with higher plasma TNF-, IFN-{gamma} and CCL2 and increased mitochondrial fission, these phenotypes were the opposite in aging CgA-KO mice. CgA-KO mice also showed increased expression of mitochondrial and nuclear-encoded complex I genes, implying that they were healthier than WT mice. Most intriguingly, the hypertension in CgA-KO mice was spontaneously reversed with aging. Supplementation of CgA-KO mice with pancreastatin, a hyperglycemic peptide produced from CgA by proteolysis, increased both blood glucose levels and blood pressure, implicating hyperglycemia, and hypertension. We conclude that age-related insulin resistance and hypertension are caused by CgA.

physiology↗

bioRxiv2021

Dynamic association of IκBα to chromatin is regulated by acetylation and cleavage of histone H4

I{kappa}Bs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for I{kappa}B homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated I{kappa}B (pS-I{kappa}B) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied. We here show that serine 32-36 phosphorylation of I{kappa}B favors its binding with nucleosomes and demonstrated that p-I{kappa}B association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-I{kappa}B binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-I{kappa}B chromatin binding and impaired goblet cell differentiation, comparable to I{kappa}B deletion. Together our results indicate that dynamic binding of I{kappa}B to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-I{kappa}B at specific stem cell compartments.

molecular biology↗