Biology subjects

Sahu, S.

Publications and source records attributed to Sahu, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond Direct Binding: Serotonin Rewires Model Brain Plasma Membrane-Mediated Communication Pathways in Y4R

Serotonin, an endogenous neurotransmitter, modulates pancreatic polypeptide (PP) recognition by the neuropeptide Y4 receptor (Y4R), a mechanism relevant to altered serotonergic signaling associated with metabolic disorder and anxiety. Although recent experiment demonstrates serotonin modulated PP dissociation kinetics of Y4R through membrane allostery, the molecular mechanism of serotonin induced lipidomic reorganization of the brain plasma membrane (BPM) relevant for Y4R activation is unknown. To this end, a total of 825.9 ns long all atom molecular dynamics simulations at 310 K elucidate the concentration-dependent coupling between serotonin partitioning, selective lipid reorganization, and PP binding and Y4R activation relevant intra-receptor communication in a heterogeneous, asymmetric model BPM. In the absence of serotonin, Y4R preferentially associates with the anionic lipids. Serotonin preferentially localizes at the lipid water interface over the PP binding and activation-associated regions of Y4R. At 6 mM, serotonin preferentially contacts phosphocholine species depleted from Y4R, whereas at 50 mM, phosphatidylinositol 4,5 bisphosphate dominates both microenvironments. These changes align with the selective remodeling of lipid networks, enhanced lipid chain ordering, and modest bilayer compaction. Weighted implementation of suboptimal paths analysis reveals concentration-dependent reorganization of communication pathways between experimentally implicated PP binding and activation associated Y4R residues. Collectively, these findings connects serotonin dependent selective lipid redistribution to altered intramolecular communication between Y4R residues associated with PP binding and G-protein coupling, and provide a framework for future experiments investigating membrane-mediated neuromodulation and neuronal signaling. These findings provide a basis for exploring lipid-targeted pharmacology as a complementary strategy to modulate GPCR allostery by altering membrane composition rather than directly targeting the receptor.

biophysics↗

bioRxiv2026

Surface Binding Sites Orchestrate Long-Range Control of Active-Site Dynamics and Glucose Tolerance in β-glucosidase, BglB

{beta}-Glucosidases are essential for lignocellulosic biomass conversion, yet their industrial utility is limited by inhibition from glucose, the reaction product. Although glucose inhibition has been extensively documented, its molecular basis remains poorly understood. Here, we demonstrate that glucose inhibition in the GH1 {beta}-glucosidase BglB from Paenibacillus polymyxa is mediated by multiple surface-exposed secondary binding sites (SBSs) that function as regulatory elements rather than passive glucose-binding patches. Kinetic analyses revealed a mixed mode of inhibition, with modest activation at low glucose concentrations followed by progressive inhibition at higher concentrations. Glucose association occurred through multiple SBSs in a concentration-dependent manner and induced localized rigidification, particularly at gatekeeper regions surrounding the active-site entrance. These surface interactions propagated through long-range coupling pathways, resulting in structural, energetic, and residue interaction network reorganization that reshaped the catalytic pocket. Functional interrogation of representative SBSs revealed distinct roles for individual sites. Mutation of a gatekeeper-associated SBS reduced local glucose association, increased glucose tolerance by more than 30%, and improved substrate affinity by approximately 38%, whereas disruption of a distal SBS compromised structural integrity and soluble protein production. Together, these findings establish a direct link between surface glucose recognition and active-site regulation, providing, to our knowledge, the first integrated evidence for the existence and functional significance of secondary glucose-binding sites in {beta}-glucosidases. More broadly, this work identifies surface SBSs as promising targets for engineering glucose-tolerant and catalytically robust enzymes for biomass conversion and related biotechnological applications.

biophysics↗

bioRxiv2026

BIRD-Seq: B2 Protein Integrated End-to-End Pipeline for dsRNA Detection and Nanopore Sequencing for Virus Monitoring

Double-stranded RNA (dsRNA) is a near-universal hallmark of active viral infection. Despite its role as a pan-viral replication intermediate, dsRNA-centred technologies for virus monitoring remain scarce and largely rely on monoclonal antibody-based approaches that, while highly sensitive, are costly and difficult to engineer, scale, or integrate with downstream assays. Here, we present a modular and antibody-free pipeline for quantitative and qualitative dsRNA analysis built around an engineered B2 protein from Flock House virus with nanomolar-range binding affinity. The pipeline is compatible with absorbance- or luminescence-based measurement formats. In a sandwich assay configuration (Sand-BIRD), sub-ng mL-1 quantification of dsRNA is achieved, comparable to the gold standard J2 monoclonal antibody, directly from crude biological samples without RNA extraction. Sand-BIRD reliably detects viral infection in both plant samples (Tomato bushy stunt virus and Grapevine fanleaf virus) and mosquitoes (West Nile virus and Dengue virus) with commercial-grade reliability. dsRNA eluted from positive samples were further processed directly by Oxford Nanopore direct sequencing, enabling identification of virus species without prior sequence knowledge or total RNA extraction. Together, this work establishes an end-to-end, sequence-agnostic workflow for direct RNA-duplex quantification and sequencing (BIRD-Seq), which has compelling potential for emerging infectious disease surveillance and next-generation point-of-care (PoC) diagnostics. Technology ReadinessBIRD-Seq is an integrated pipeline for agnostic dsRNA detection and sequencing designed for broad-spectrum virus monitoring. A Technology Readiness Level (TRL) 5 under NASAs classification framework has been reached as BIRD-Seq has been validated in laboratory-relevant environments using real-world samples, including virus-infected plants and mosquitoes. The ELISA-based sensing platform employs engineered variants of the B2 protein (from Flock House virus) in a sandwich assay format for dsRNA capture and detection, achieving sensitivity comparable to the gold-standard J2 monoclonal antibody. Unlike traditional antibody-based methods, the B2 protein offers key practical advantages: straightforward production in bacterial expression systems, high versatility, and reduced manufacturing costs, as well as direct compatibility with crude extract monitoring, eliminating the need for RNA extraction. Captured B2/RNA duplexes can then be directly eluted from the ELISA microplates and subjected to downstream nanopore direct RNA sequencing, providing both quantitative and qualitative information on the underlying virus infection, a capacity enabled by the near-universal nature of dsRNA as a pathogen-associated molecular pattern. That said, further validation on large-scale field-collected and clinical samples will be essential before widespread deployment can be envisioned. While the B2 sandwich assay offers favorable cost-efficiency over antibody-based alternatives, the relatively high cost of Oxford Nanopore direct RNA sequencing remains an important economic constraint. Nevertheless, the growing importance of dsRNA detection across virus sensing, mRNA vaccine development, innate immunity research, and human disease diagnostics, combined with the increasing role of portable long-read sequencing in emerging infectious disease (EIDs) surveillance, positions BIRD-Seq as an innovative and competitive diagnostic platform. Highlights- Double-stranded RNA (dsRNA) is one of the critical pathogen-associated molecular patterns for viral invasion in the host. A protein-based sandwich assay for dsRNA detection in crude biological samples with a sub-ng mL-1 order detection limit was developed, achieving similar sensing efficiency in comparison to expensive and proprietary monoclonal antibody-based ELISA methods. - The quantitative detection of RNA duplex is coupled with an Oxford nanopore direct dsRNA sequencing method for virus species identification and qualitative analysis. - This is one of the very first dsRNA-centered end-to-end workflows for virus monitoring and sequencing, validated for both infected plant and animal samples.

bioengineering↗

bioRxiv2026

Dual inhibition of GTP-bound (ON) and GDP-bound (OFF) KRASG12C suppresses PI3Kα and leads to potent tumor inhibition

Current approved KRASG12C inhibitors covalently bind the inactive GDP-bound (OFF) form of KRASG12C. Recently, KRASG12C inhibitors that selectively bind to the GTP-bound (ON) form of both KRASG12C (ON) and (OFF) forms have been reported and entered clinical testing. In principle, KRASG12C (ON) inhibitors may be less susceptible to adaptive mechanisms that promote resistance to (OFF) inhibitors, however the specific mechanisms that differentiate the activity of (ON) versus (OFF) inhibition are not well understood. We profiled the activity of BBO-8520, a covalent dual inhibitor of GTP-bound (ON) and GDP-bound (OFF) KRASG12C, in KRASG12C-mutant non-small cell lung cancer models. BBO-8520 exerted more potent and sustained inhibition of KRASG12C and anti-tumor activity in vitro and in vivo compared with sotorasib, a KRASG12C (OFF)-only inhibitor. While cells treated with BBO-8520 or sotorasib both exhibited feedback reactivation of MAPK signaling driven by wild-type HRAS/NRAS isoforms, more durable suppression of KRASG12C by BBO-8520 was associated with decreased PI3K-AKT activation in vitro. Disruption of the interaction between RAS and PI3K using a novel protein:protein interaction inhibitor suppressed PI3K-AKT activation and increased the tumor response to sotorasib to a similar level as BBO-8520. Moreover, in some contexts, disruption of RAS-PI3K further increased the anti-tumor activity of BBO-8520 monotherapy. These results reveal mechanistic differences between KRAS (ON) and (OFF) inhibitors, highlight the importance of PI3K-AKT signaling in driving resistance to KRAS inhibition in lung cancer, and suggest combination strategies that suppress PI3K-AKT to improve the response to KRAS inhibitors.

cancer biology↗

bioRxiv2026

A single BRCA2 BRC repeat supports viability while multiple repeats ensure resilience under stress

BRC repeats are integral to BRCA2 function and mediate RAD51 loading during homologous recombination (HR). Their number varies across species, but mammals, including mice and humans, harbor eight repeats. Prior studies have suggested functional redundancy among these repeats, but the biological significance of maintaining multiple repeats remains unresolved. Here, we demonstrate that the presence of a single BRC repeat, either BRC2 or BRC4, is sufficient for mouse embryonic stem cell (mESC) viability, RAD51 loading, PARP inhibitor resistance and protection of stalled replication forks. Consistent with these findings, we show that knock-in mice with a single BRC repeat 2 or 4 are viable and exhibit normal growth and fertility. In contrast, embryonic fibroblasts from these mice display genomic instability and impaired RAD51 recruitment. Notably, this defect is rescued under low oxygen culture conditions, which mimics physoxic levels, whereas exposure to oxidative stress impairs RAD51 recruitment in mESCs harboring a single BRC repeat. Together, our findings indicate that while a single BRC repeat is sufficient under physiological conditions, the evolutionary retention of multiple BRC repeats likely ensures robust genome stability under extreme oxidative stress.

genetics↗

bioRxiv2025

Domain Compatibility and Linker Design Dictate the Success of Chimeric Cellulase Engineering

Efficient conversion of lignocellulosic biomass into fermentable sugars remains a major challenge due to individual cellulases limited synergy and catalytic efficiency. Engineering chimeric enzymes provides a promising strategy to streamline biomass hydrolysis by combining complementary catalytic activities in a single protein, thereby enhancing efficiency and lowering process costs. In this study, we constructed chimeric cellulases by fusing a thermophilic GH1 {beta}-glucosidase (TsBG) with endoglucanases from the GH5 (BsEG2) or GH9 (BlEG) families through flexible peptide linkers. Constructs containing BsEG2 exhibited a pronounced loss of {beta}-glucosidase activity and reduced endoglucanase activity, whereas substitution with the full-length BlEG restored dual functionality under identical design conditions. The optimized chimera (BlEG+(G4S)2+TsBG) demonstrated enhanced catalytic performance, with a 4.8-fold lower Km, a 1.7-fold higher Vmax, and an increased kcat (from 1088 to 1454 s-1). The chimera also exhibited enhanced stability, retaining [~]10 % higher activity under elevated cellobiose (up to 300 mM) and >90 % specific activity in 2.5 M NaCl. Molecular dynamics simulations further revealed that activity loss in non-optimized constructs arose from C-terminal structural instability and steric clashes, underscoring the critical role of domain orientation and linker flexibility in chimera design. These findings establish a chimeric cellulase that integrates endoglucanase and {beta}-glucosidase activities in a single polypeptide, offering a robust and cost-effective biocatalyst for lignocellulosic biomass conversion.

biochemistry↗

bioRxiv2025

Affinity-matured CD72-targeting Nanobody CAR T-cells Enhance Elimination of Antigen-Low B-cell Malignancies

BackgroundChimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. Here, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models. MethodsPrimary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al.1 CAR T-cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line- or patient-derived xenografts implanted in NOD scid gamma (NSG) mice. ResultsWe first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into chimeric antigen receptor (CAR) T-cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T-cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for scFv-based CAR T-cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody. ConclusionsTogether, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22. What is already known on this topicPrevious work using single chain variable fragment (scFv) based CAR Ts has suggested that improving affinity for target antigen could potentially help mitigate tumor resistance mediated by antigen downregulation, or baseline low antigen density. However, it is unknown whether this same dynamic holds for CAR T-cells that utilize different antigen recognition elements, such as nanobodies. What this study addsHere we show that affinity maturation of nanobody-based CAR T-cells (nanoCARs) targeting CD72 can improve their in vitro efficacy versus CD72-low tumors; however, in vivo efficacy differences are more modest. Furthermore, we show that for refractory B-cell malignancies, surface CD72 appears preserved after CD19 resistance even in situations where CD22 is strongly downregulated. How this study might affect research, practice or policyCD72 warrants further investigation as a preferred immunotherapy target in the context of CD19-refractory B-cell cancers, though nanobody affinity maturation is not a universal solution to the challenge of low tumor surface antigen density.

immunology↗

bioRxiv2024

Translation components in adult Drosophila melanogaster adipocytes regulate the ovarian germline stem cell lineage

Adult stem cells, which support tissue homeostasis and damage repair, are influenced by whole organism physiology. Dietary input has a major impact on the stem cell supported ovary in Drosophila melanogaster females, appropriately matching reproductive output to nutrient availability. Previous work has shown that inter-organ communication plays a role in modulating the ovarian response to diet. Specifically, amino acid sensing by the adipose tissue remotely controls germline stem cells and their progeny. While we have shown that activation of the amino acid response pathway, a part of the integrated stress response, and mTOR signaling in adipocytes impacts germline stem cell maintenance and ovulation, it is unclear how downstream signaling mediates these responses. Here, using a combination of genetic and cell biological tools, we show that regulation of translation in adult adipocytes impacts the ovarian germline stem cell lineage, from stem cell maintenance to ovulation of mature oocytes. This work strongly suggests that the adipose tissue produces specific factors to control stem cell activity in the ovary and highlights how inter-organ communication underlies organismal physiological responses to diet.

physiology↗

bioRxiv2024

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

bioengineering↗

bioRxiv2024

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations. Statement of significanceUsing a new in silico strategy we reveal counteracting determinants of patient survival in Acute Myeloid Leukemia that co-opt well-defined mutation-dependent splicing programs. Broad poor-prognosis splicing and leukemia stem cell survival could be rescued through pharmacological inhibition (PRMT5) or target deletion (IRAK4), opening the door for new precision therapies. Competing InterestsConflict-of-interest disclosure: DTS. serves on the scientific advisory board at Kurome Therapeutics; is a consultant for and/or received funding from Kurome Therapeutics, Captor Therapeutics, Treeline Biosciences, and Tolero Therapeutics; and has equity in Kurome Therapeutics. AV has received research funding from GlaxoSmithKline, BMS, Jannsen, Incyte, MedPacto, Celgene, Novartis, Curis, Prelude and Eli Lilly and Company, has received compensation as a scientific advisor to Novartis, Stelexis Therapeutics, Acceleron Pharma, and Celgene, and has equity ownership in Throws Exception and Stelexis Therapeutics.

cancer biology↗

bioRxiv2023

AVENGERS: Analysis of Variant Effects using Next Generation sequencing to Enhance BRCA2 Stratification

Accurate interpretation of genetic variation is a critical step towards realizing the potential of precision medicine. Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants. Due to limited clinical, familial and/or epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). To determine the functional impact of VUSs, here we develop AVENGERS: Analysis of Variant Effects using NGs to Enhance BRCA2 Stratification, utilizing CRISPR-Cas9-based saturation genome editing (SGE) in a humanized-mouse embryonic stem cell line. We have categorized nearly all possible missense single nucleotide variants (SNVs) encompassing the C-terminal DNA binding domain of BRCA2. We have generated the function scores for 6270 SNVs, covering 95.5% of possible SNVs in exons 15-26 spanning residues 2479-3216, including 1069 unique missense VUS, with 81% functional and 14% found to be nonfunctional. Our classification aligns strongly with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. Our statistical classifier exhibits 92.2% sensitivity and 96% specificity in distinguishing clinically benign and pathogenic variants recorded in ClinVar. Furthermore, we offer proactive evidence for 617 SNVs being non-functional and 3396 SNVs being functional demonstrated by impact on cell growth and response to DNA damaging drugs like cisplatin and olaparib. This classification serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counselors assessing BRCA2 VUSs in patients.

genetics↗

bioRxiv2023

Protein condensate fusion mapping using Hippopede curve

Protein and nucleic acid condensation in cells has been a major focus of biophysical studies in recent years. Such droplets, condensates, or membraneless cellular organelles that form by condensation have been shown to behave like liquid or viscoelastic fluids. In vitro reconstitution of condensates of purified protein and nucleic acid have been used to better understand the phenomena, specifically to quantify the material properties of these condensates. One of the signature properties of a liquid-like condensate is the fusion of two round droplets into a larger droplet. Quantitative imaging and analysis of the fusion process can report the biophysical properties of the condensates, such as surface tension and viscosity in a non-invasive way. Here we take advantage of a model in soft matter physics, hippopede curves, to the condensate fusion problem to estimate these biophysical properties directly and compare the results with existing models in the field. In addition, this method approximates additional parameters such as the surface area and surface curvatures during the fusion process over time.

biophysics↗

bioRxiv2023

Protein Assembly Modulation: A New Approach to ALS Therapeutics

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with a complex, multifactorial pathophysiology, most commonly manifest as loss of motor neurons. We introduce a new mechanism of ALS pathogenesis via a novel drug-like small molecule series that targets protein disulfide isomerase (PDI) within a previously unappreciated transient and energy-dependent multi-protein complex. This novel drug was found to have activity in cellular models for both familial and sporadic ALS, as well as in transgenic worms, flies, and mice bearing a diversity of human genes with ALS-associated mutations. These compounds were initially identified as modulators of human immunodeficiency virus (HIV) capsid assembly in cell-free protein synthesis and assembly (CFPSA) systems, with demonstrated antiviral activity in cell culture. Their advancement as ALS-therapeutics, and the subsequent separation of activity against HIV and ALS in chemical subseries through structure-activity-relationship optimization, may provide insights into the molecular mechanisms governing pathophysiology of disordered homeostasis relevant to ALS.

neuroscience↗

bioRxiv2023

Identifying Selectivity Filters in Protein Biosensor for Ligand Screening

Specialized sensing mechanisms in bacteria enable the identification of cognate ligands with remarkable selectivity in highly xenobiotic-polluted environments, where these ligands are utilized as energy sources. Here, via an integrated all-atom computer simulation, biochemical assay and isothermal titration calorimetry approaches we determine the molecular basis of MopR, a phenol biosensors complex selection process of ligand entry. Our results reveal a set of strategically placed selectivity filters along the ligand entry pathway of MopR. These filters act as checkpoints, screening diverse aromatic ligands at the protein surface based on their chemical features and sizes. Ligands meeting specific criteria are allowed to enter the sensing site in an orientation-dependent manner. Sequence and structural analyses demonstrate the conservation of this ligand entry mechanism across the sensor class, with individual amino acids along the selectivity filter path playing a critical role in ligand selection. Together, this investigation highlights the importance of interactions with the ligand entry pathway, in addition to interactions within the binding pocket, for achieving ligand selectivity in biological sensing. The findings enhance our understanding of ligand selectivity in bacterial phenol biosensors and provide insights for the rational expansion of the biosensor repertoire, particularly for the biotechnologically relevant class of aromatic pollutants.

biochemistry↗

bioRxiv2023

Iron chelation by deferoxamine as useful adjunct therapeutics in murine tuberculosis.

Dysregulated iron metabolism is reported in tuberculosis patients; therefore, it represents an opportunity for developing host-directed therapeutics. This study monitored the antimycobacterial properties of an iron chelator, i.e., Deferoxamine (DFO/D), and its impact on the transcript and metabolite levels of Mycobacterium tuberculosis (Mtb) in vitro. For in vivo validation, a group of mice received ferric carboxymaltose to create an iron overload condition, and controls were aerosol-infected with Mtb H37Rv. Mtb-infected mice received isoniazid (INH/H) and rifampicin (RIF/R) in combination with or without DFO before tissue-specific CFU assay, liver metabolite screening and iron quantification using mass spectrometry. DFO showed antimycobacterial properties comparable to INH in vitro. DFO treatment deregulated (log2DFO/control>{+/-}1.0) Mtb transcript (n=137) levels, the majority of which encode for iron-containing proteins and proteins involved in stress response. DFO treatment up-regulated Rv3622c (PE32), Rv2353c (PPE39) and Rv3022A (PE29) genes and conditional knocking down of ABC transporter like irtA by anhydrotetracycline (Atc) inducible CRISPR interference (CRISPRi) approach compromised Mtb growth showing their potential involvement in iron metabolism. Global Mtb metabolite analysis using GC-MS identified a set of 5 deregulated metabolites indicating a perturbed pentose phosphate pathway and inositol phosphate metabolism in the host upon DFO treatment. Iron-overloaded mice exhibited significantly higher tissue mycobacterial burden at two weeks post-infection, and the efficacy of INH and RIF were compromised, corroborating with previous reports. Iron chelation by DFO or combined with/adjunct to RIF and INH significantly reduced the lung/tissue mycobacterial burden at four weeks post-treatment, specifically in the first ([~]0.5 log) and second weeks ([~]0.5 log) of treatment. The intracellular pro-inflammatory cytokine levels in the lung CD4+ T cells of INH and RIF-treated groups with or without DFO were similar, suggesting DFO has a direct role in Mtb survival and metabolism rather than improved infection, and the efficacy of INH and RIF were compromised, corroborating with previous reports. DFO adjunct to RIF and INH treatment significantly altered liver arginine biosynthesis, which directly neutralizes ammonia and is immune-supportive. Conventionally, DFO is used for treating acute iron toxicity that is common in thalassemic patients, and this study demonstrates DFO has potential as adjunct therapeutics for tuberculosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/543389v3_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@5066fdorg.highwire.dtl.DTLVardef@99a292org.highwire.dtl.DTLVardef@80426dorg.highwire.dtl.DTLVardef@892436_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1-mutant lung cancer

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+ degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)--key downstream effectors of LKB1-- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCELoss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

cancer biology↗

bioRxiv2023

Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

Intracellular Salmonella resides and multiplies in cholesterol-rich specialized compartment called Salmonella-containing vacuoles (SCVs) and avoids fusion with acidic lysosomes. Given, lysosomes are primary organelle that redistributes LDL derived cholesterol to other organelles; we questioned how lysosomal cholesterol can be transported to SCV. We demonstrate here that peroxisomes are recruited to SCVs in human primary macrophages, epithelial cells and functions as pro-bacterial organelles. Further, this interaction is assisted by SseI, a Salmonella effector protein containing mammalian peroxisome targeting sequence. SseI localizes to peroxisome, interacts and activates a host Ras GTPase, ARF-1 on the peroxisome membrane. Activation of ARF-1 leads to recruitment of phosphatidylinsolitol-5- phosphate-4 kinase to generate phosphatidylinsolitol-4-5-bisphosphate on peroxisomes. Accordingly, the{Delta} sseI strain showed reduced virulence in cell lines and during mice infection. Taken together, our work identified a fascinating mechanism by which a pathogen targets host organelles via its secretory effectors and exploits host metabolic intermediates for its intracellular proliferation.

cell biology↗

bioRxiv2022

Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates

Microtubule organization in cells is essential for the internal structure and coordination of events of intracellular transport, mitosis, and cell motility. For many cell types, microtubule organization is dominated by centrosomal nucleation that use gamma-tubulin to template filaments. Yet, some cell types lack centrosomes or centrioles, such as plant cells. Instead, microtubules nucleate from regions with high concentrations of microtubule binding and nucleating proteins. A mechanism that can drive high local concentrations of nucleators is liquid-liquid phase separation of proteins with intrinsically disordered regions. Here, we report that the plant microtubule nucleator and crosslinking protein, MAP65-1, can form phase separated condensates at physiological salt and temperature without extra crowding agents. These condensates are liquid at first and can mature to gel-like phases over time and with different environmental conditions. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate. Condensate-induced cytoskeletal formation could be a universal mechanism for organization of the microtubule and actin cytoskeletons in all cell types, especially cells without centrosomes.

biophysics↗