Biology subjects

Bera, S.

Publications and source records attributed to Bera, S..

18 recordsLinked to original sources

bioRxiv2026

Structure-activity of outer membrane proteins in native bacterial membrane vesicles by solid-state NMR

The outer membranes (OMs) of bacterial pathogens are potent virulence factors and serve as the first line of defense against host immunity. Their striking bilayer asymmetry is essential for function but poses an exceptional challenge for reconstitution in vitro, limiting structure-activity analysis to artificial non-native platforms that can interfere with structure and function. Here, we describe bacterial OM vesicles (OMVs), natively secreted from the cellular OM during bacterial cell growth and development, as an effective vehicle for structure-activity analysis in situ based on solid-state nuclear magnetic resonance (NMR). We show that E. coli OMVs may be engineered to express a range of isotopically labeled target OM proteins, and isolated for solid-state NMR magic angle spinning (MAS) experiments. High resolution NMR spectra are obtained for three bacterial virulence factors: the adhesion invasion locus (Ail) and plasminogen activator protease (Pla) from Yersinia pestis, and the major porin (OmpF) from E. coli. The spectra reflect the native protein structures, report on the specific OMV membrane environment, and may be used to map protein interactions with their human host ligands, specifically the multifunctional glycoprotein Vitronectin (Vn) which binds Ail as part of its serum protection activity. Notably, OMVs support protein functionality, enabling structure and activity to be correlated in situ. OMVs expressing plasmid-encoded Ail recruit human Vn and confer serum protection to wild-type E. coli cells, while OMVs expressing plasmid-encoded Pla support the proteolytic activity of Pla. Taken together, the data establish OMVs as a robust new platform for structure-activity analysis of OM proteins in situ, offer new insights about the complexity of the bacterial OM, and reveal additional functional aspects of OMVs as key ancillary units of bacterial infection.

biophysics↗

bioRxiv2026

Breast cancer stem cells mediated CD8+ T cell exhaustion among different molecular subtypes of breast cancer regulated via NOTCH1/RBPJ/PD-L1 axis

BackgroundBreast cancer stem cells (BCSCs) contribute significantly to breast cancer (BC) mortality among women globally. It underpins tumor heterogeneity in BC by driving variations in stemness potential and altering immune microenvironment. However, how BCSCs, subpopulations of breast cancer cells from distinct molecular subtypes differentially modulate CD8 T-cell exhaustion and immune dysfunction remain unclear. MethodsWe conducted our study from patients with BC of four subtypes. MACS sorted (Lin-CD44+CD24-) BCSCs were prepared for mammosphere formation assay from mastectomies samples. Flow-cytometry was used to analyze breast cancer stem cells (BCSCs). Immunofluorescence, immunohistochemistry, Real Time and Reverse Transcriptase PCR array, Chromatin-immunoprecipitation assay, Transwell, ELISA, Western blotting, Cloning, Transfection, Knockdown, chromatin immunoprecipitation approaches were used to investigate the underlying mechanisms. ResultsHere, we report that BCSCs actively participate in tumor progression by modulating effector CD8 T-cells. Triple-negative breast cancer (TNBC), being the subtype with the most adverse outcomes, sustains the enrichment of stem cell regulating transcription factors like NANOG, OCT4 and SOX2 compared to HER2, Luminal B, and Luminal A subtypes. Tumor from TNBC patients exhibited an exhausted phenotype within CD8 T-cell infiltrates with PD1high TIM3high LAG3 high IFN{gamma}low signature. BCSCs induced increased proportion of exhausted CD8 T-cells, predominantly in the TNBC subtype. Cell-surface Notch1 expression was upregulated in BCSCs across all molecular BC subtypes, with the highest elevation observed in TNBC. Knockdown or inhibition of Notch1 downregulated stemness-associated genes and diminished CSC-mediated induction of CD8 T-cell exhaustion. Cumulatively, these findings suggest that assessment of high Notch1 and Nanog frequency within BCSCs can guide Notch1-targeted therapies and may formulate for new combinatorial treatment strategies to improve patient outcomes. Additionally, therapeutic targeting of BCSC-intrinsic NOTCH1-NANOG/NOTCH1-PD-L1 axis could represent an effective strategy to reduce stemness programs and alter BCSC-driven CD8 T-cell exhaustion, majorly in aggressive subtypes such as TNBC. ConclusionsBCSCs aggressiveness is perpetuated through Notch1-mediated axis. Targeting Notch1 would reduce stemness (majorly NANOG), survival, as well as prevent CD8 T-cell exhaustion (upregulating PD-1, TIM3, LAG3), thereby weakening tumor progression.

cancer biology↗

bioRxiv2025

Delineation of a novel assembly intermediate in retroviral integration pathway

Retroviral integration is mediated by viral integrase (IN) which synapses two viral long terminal repeat (LTR) DNA ends and produces a series of nucleoprotein complexes known as intasomes. While structural studies of mature intasomes have illuminated key aspects of their architecture and provided insights into the integration reaction, the sequence of events driving IN oligomerization and engagement of the viral DNA pairing remains unclear. Here, using complementary biochemical and biophysical approaches, including ensemble and single molecule FRET, we reveal that integration progresses through a key transient intermediate that leads to the mature intasome. We demonstrate that Rous Sarcoma Virus (RSV) intasome assembly pathway proceeds through a tetrameric intermediate where two IN dimers engage a single DNA end. This complex subsequently oligomerizes to form mature functional octameric intasome in which two DNA ends are juxtaposed for concerted integration. These findings provide mechanistic insights into the stepwise pathway of retroviral integration and define a previously uncharacterized intermediate critical for intasome maturation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/675266v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@57a82aorg.highwire.dtl.DTLVardef@e17110org.highwire.dtl.DTLVardef@c5f4c4org.highwire.dtl.DTLVardef@4886d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Genome sequencing, de novo assembly and annotation of the commercially important bamboo, Bambusa tulda Roxb.

Bambusa tulda Roxb., a member of the Bambusoideae subfamily, is an ecologically and commercially important plant resource widely distributed on the Indian subcontinent. Our study reports long-read PacBio HiFi sequencing and genome assembly of B. tulda. Flow cytometry analysis revealed its estimated genome size [~]3 Gb. The de novo genome assembly of B. tulda predicted 43 contigs, distributed across three subgenomes, with a total size of 1.37 Gb, contig N50 of 35.69 Mb, and BUSCO score 99%. Repetitive elements constitute 63.31% of the genome. Functional annotation predicted 56,890 protein-coding genes, constituting 19.44% of the genome. This high-quality draft genome assembly will serve as an invaluable resource for future studies on the life history traits, phylogenomic analysis, comparative genomics, and targeted genome modification for important trait improvement of B. tulda.

genomics↗

bioRxiv2025

Comparative evaluation on the fermentative potential of single and assorted fruit and vegetable waste for production of bioethanol

Fruit and vegetable waste (FVW) constitute a large part of municipal solid waste (MSW) and create significant environmental challenges by emitting the greenhouse gas methane. In contrast, these are a big source of fermentable sugar to produce bioenergy. Therefore, in this study, the Scanning Electron Microscopy (SEM), Attenuated total reflectance-fourier-transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and biochemical quantification of -cellulose and acid-insoluble lignin contents were carried out to compare fermentative potential between single (potato peel vs. orange peel) and assorted vegetable and fruit wastes. Collectively, the study indicates that assorted vegetable waste holds enormous biofuel potential by having low moisture content (6.78 %), moderately low decomposition temperature (Tmax: 299.36 {degrees}C), low crystallinity index (CrI: 19.12%), and high -cellulose contents (32.35 - 47.73 gm%) yielding high sugar (325.65 mg/g). The study also optimized several key fermentation parameters such as pH, sugar concentration and the time to produce bioethanol. It was observed that maximum production of ethanol (2.14%) was achieved at 28{degrees}C, at pH 6.3 with a higher initial sugar concentration of 200mg/g by using Saccharomyces cerevisiae strain NCIM 3594. This study supports the likely utility of unsorted vegetable waste to obtain bioethanol at a mass scale.

plant biology↗

bioRxiv2025

Tumor-Educated-Platelets interact with Breast Cancer-Stem-Cells via P-selectin- PSGL1 and ensure stemness and metastasis through WNT-β-Catenin-VEGF-VEGFR2 intra-cellular signaling: Therapeutic modulation by aspirin

BackgroundProtagonistic role of platelets promote capillary infiltration of tumors for distant metastasis along with immunosurveillance. Despite existing reports highlighting role of platelets in tumorigenesis, its impact on breast cancer stem cells (BCSCs) remain underexplored. Our first ever report on murine and human system, accentuate that, tumor educated platelets (TEPs) of luminal A and TNBC subtypes are distinct from healthy counterparts, collaborating with BCSCs to generate sub-variants that elevate tumor aggressiveness. MethodsImpact of TEPs on BCSCs was evaluated from primary breast tumor and blood samples of luminal A/TNBC patients along with EC/4T1 murine breast tumor models and MCF-7/MDA-MB-231 cell lines. For downstream assays, TEPs were co-cultured with breast tumor samples or cell lines, followed by magnetic sorting of lin-CD44+CD24- BCSCs. TEP induced alterations of BCSCs were evaluated from 3D tumorsphere, colony formation, transwell migration, scratch-wound healing, matrigel invasion, in-vitro tube formation assays. Fluorescence-confocal microscopy, RT-PCR, flow-cytometry, western-blotting was utilized to decipher the role of genes and protein involved in stemness, metastasis along with the transcription factors in the downstream signaling cascade, followed by verifications by RNAi. ResultsTEPs have elevated expression of P-selectin and interacts with BCSCs via P-selectin and PSGL1 on BCSCs surface. Treatment with aspirin had restorative impact on P-selectin level, converting TEPs from active to resting platelet (RP) state. Under TEPs influence, BCSCs were tumorigenic, clonogenic, multidrug resistant, invasive with numerous invadopodia and remained skewed towards mesenchymal phenotype. Administration of RP reduced TEP associated BCSC virulence both in-vivo and in-vitro. P-selectin-PSGL1 interaction results in binding of WNT to FRIZZLED followed by stabilization and nuclear translocation of {beta}-Catenin. Nuclear {beta}-Catenin promotes stemness-EMT (Epithelial to mesenchymal transition)- metastasis, along with stimulation of autocrine VEGF-VEGFR2 cascade. Inhibition of WNT and VEGFR2 by RNAi confirmed the critical role of this axis in regulating TEPs influence on BCSCs. ConclusionThese insights into TEPs-BCSC interplay, acknowledges TEPs, as-well-as unveils novel receptor-ligand signaling cascade between TEPs and BCSCs, that could be a beneficial therapeutic strategy to target cancer metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/657784v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@11db5d0org.highwire.dtl.DTLVardef@18b7270org.highwire.dtl.DTLVardef@64c2borg.highwire.dtl.DTLVardef@172d68f_HPS_FORMAT_FIGEXP M_FIG C_FIG Cellular interaction between TEPs P-selectin and BCSCs PSGL1 to regulate stemness, EMT, metastasis and angiogenesis via WNT-{beta}-Catenin-VEGF-VEGFR2 cascade: Modulation by pharmacological inhibitor and RNAi.

cancer biology↗

bioRxiv2025

Molecular and biological characterization of a distinct species of Lolavirus infecting different accessions of seashore paspalum, a turfgrass, widely grown in the United States

Seashore paspalum (Paspalum sp.), is an economically significant grass used in golf courses, sports fields, and landscaping in the United States. A novel Lolavirus, tentatively named paspalum latent virus (PaLV), was identified for the first time in seashore paspalum plants from the USDA National Plant Germplasm System (NPGS) using high-throughput sequencing. Three complete genome sequences of PaLV from different Paspalum accessions, with a length of 6,995 nucleotides (nt), not including the poly(A) tail, were obtained by Rapid Amplification of cDNA Ends and Sanger sequencing. Phylogenetic analysis based on the replicase protein sequences from the Alphaflexiviridae family revealed that PaLV grouped with the Lolavirus genus, with the closest relative being Lolium latent virus (LoLV). PaLV shares less than 72% nt identity to the replicase and coat protein genes of LoLV, which demarks PaLV as a new species and the second member of the genus. Furthermore, the coat protein region showed intense negative selection pressure and low spatially structured diversity. Host range analysis of PaLV showed that wheat, corn, sorghum, and Lolium are systemic hosts of PaLV. A one-step RT-PCR technique was developed to reliably detect PaLV infection.

pathology↗

bioRxiv2025

Specialized Molecular Pathways Drive the Formation of Light-Scattering Assemblies in Leucophores

Pigmentation plays a vital role in the survival of organisms, supporting functions such as camouflage, communication, and mate attraction. In vertebrates, these functions are mediated by specialized pigment cells known as chromatophores of which, uric acid crystal-forming leucophores remain the least understood, with little known about their molecular mechanisms. A key question in pigment cell biology is whether different crystal chemistries require distinct molecular pathways, or whether similar cellular processes drive the formation of diverse crystals. This study was designed to unravel the uncharacterized process of uric acid crystallization in leucophores and compare them to guanine crystal formation in iridophores and pterin formation in xanthophores. The results of our transcriptomic, ultrastructural, and metabolomic analyses, demonstrate that leucophores share molecular pathways with iridophores, particularly those connected to organelle organization and purine metabolism, but express discrete genes involved in uric acid biosynthesis and storage. Additionally, leucophores share intracellular trafficking and pterin biosynthesis genes with xanthophores, suggesting universally conserved processes. Ultrastructural studies reveal star-like fibrous structures in leucosomes, which likely serve as scaffolds for unique one-dimensional uric acid assemblies that radiate from the core and act as efficient light scatterers. These findings provide new insights into leucophore cell biology and the specialized mechanisms driving molecular crystalline assembly, and reveal that while some cellular processes are conserved, the specific chemistry of each crystal type drives the evolution of distinct molecular pathways.

cell biology↗

bioRxiv2024

Experimental type 1 diabetes metabolically rejuvenates CD8+ T cells for improved control of tumor growth through an IGF1-IGF1R axis

AbstractEpidemiological studies suggest that patients with pre-existing type 1 diabetes (T1D) have a decreased risk of developing melanoma, prostate cancer, and breast cancer, although the underlying mechanism remains to be elucidated. In translational modelling, we observed that streptozotocin (STZ) induced T1D mice exhibited restricted melanoma and carcinoma (mammary, lung and colon) growth in association with extended overall survival. Tumor-infiltrating CD8+ T cells were found to be responsible for tumor growth restriction. Tumor infiltrating CD8+ T cells but not tumor cells themselves exhibited higher glycolytic and cytotoxic activities in T1D hosts. Such improved anti-tumor T cell function was linked to selective upregulated expression of insulin-like growth factor 1, insulin-like growth factor 1 receptor, and phospho-mTOR in CD8+ T cells in the TME. T1D patient derived CD8+ T cells displayed superior activation in vitro after tumor antigen stimulation vs. non-diabetic CD8+ T cells. Activation of T1D patient derived CD8+ T cells was sensitive to targeted antagonism of IGF1R and mTOR, supporting the operational involvement of the IGF1R-mTOR signaling axis. Our results suggest that selective activation of the intrinsic IGF1R-mTOR signaling axis in CD8+ T cells represents a preferred endpoint to achieving more effective immunotherapy outcomes and improved cancer patient management. SignificanceExperimental type 1 diabetes decelerates tumor growth through metabolic activation of cytotoxic T cells dependent on an IGF1R-mTOR signaling pathway. CD8+IGF1R+IGF1+ T cells play a crucial role in T1D dependent tumor control.

cancer biology↗

bioRxiv2024

A novel pyridoindole improves the recovery of residual hearing following cochlear implantation after a single preoperative application

Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide. Due to the heterogeneity of causes for SNHL, effective treatment options remain scarce, creating an unmet need for novel drugs in the field of otology. Cochlear implantation (CI) currently is the only established method to restore hearing function in profound SNHL and deaf patients. The cochlear implant bypasses the non-functioning sensory hair cells (HCs) and electrically stimulates the neurons of the cochlear nerve. CI also benefits patients with residual hearing by combined electrical and auditory stimulation. However, the insertion of an electrode array into the cochlea induces an inflammatory response, characterized by the expression of pro-inflammatory cytokines, upregulation of reactive oxygen species, and apoptosis and necrosis of HCs, putting residual hearing at risk. Here, we characterize the effects of the small molecule AC102, a pyridoindole, for its protective effects on residual hearing in CI. We show that AC102 significantly preserves hearing thresholds across the whole cochlea and confines the cochlear trauma to the directly mechanically injured area. In addition, AC102 significantly preserves auditory nerve fibers and inner HC synapses throughout the whole cochlea. AC102s effects are likely elicited during the inflammatory phase of electrode insertion trauma (EIT) and mediated by anti-apoptotic and anti-inflammatory properties, as uncovered by an in vitro assay of ethanol induced apoptosis and evaluation of mRNA expression of pro-inflammatory cytokines in an organotypic ex vivo model of EIT. The results in this study highlight AC102 as a promising compound for the attenuation of EIT during CI. Moreover, as the inflammatory response in cochlear implantation shares similarities to other etiologies of SNHL, a beneficial effect of AC102 can be inferred for other inner ear conditions as well.

neuroscience↗

bioRxiv2023

Molecular determinants for Rous sarcoma virus intasome assemblies

Integration of retroviral DNA into the host genome involves formation of integrase (IN)-DNA complexes termed intasomes. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate. The STC structure had an overall resolution of 3.36 [A] and 3 [A] in the conserved intasome core (CIC) region. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the CIC, similar to previously shown with the RSV octameric cleaved synaptic complex (CSC) intasome produced with IN and viral DNA only. An extensive analysis of higher-resolution STC structure helped in identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246 and S124 in CSC and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

biochemistry↗

bioRxiv2022

Mathematical modeling suggests that a single Plasmodium-specific CD8 T cell can eliminate a malaria liver stage in mice

Vaccination strategies in mice inducing high numbers of memory CD8+ T cells specific to a single epitope are able to provide sterilizing protection against infection with Plasmodium sporozoites. We have recently found that Plasmodium-specific CD8+ T cells cluster around sporozoite-infected hepatocytes but whether such clusters are important in elimination of the parasite remains incompletely understood. Here we used our previously generated data in which we employed intravital microscopy to longitudinally image 32 GFP-expressing Plasmodium yoelii parasites in livers of mice that had received activated Plasmodium-specific CD8+ T cells after sporozoite infection. We found significant heterogeneity in the dynamics of the normalized GFP signal from the parasites (termed "vitality index" or VI) that was weakly correlated with the number of T cells near the parasite. We also found that a simple model assuming mass-action, additive killing by T cells well describes the VI dynamics for most parasites and predicts a highly variable killing efficacy by individual T cells. Given our estimated median per capita kill rate of k = 0.031/h we predict that a single T cell is typically incapable to kill a parasite within the 48 hour lifespan of the liver stage in mice. Stochastic simulations of T cell clustering and killing of the liver stage also suggested that 1) three or more T cells per infected hepatocyte are required to ensure sterilizing protection; 2) both variability in killing efficacy of individual T cells and resistance to killing by individual parasites may contribute to the observed variability in VI decline, and 3) the stable VI of some clustered parasites cannot be explained by measurement noise. Taken together, our analysis for the first time provides estimates of efficiency at which individual CD8+ T cells eliminate intracellular parasitic infection in vivo.

immunology↗

bioRxiv2021

Legume plant defenses and nutrients mediate indirect interactions between soil rhizobia and chewing herbivores

Soil bacteria that form mutualisms with plants, such as rhizobia, affects susceptibility of plants to herbivores and pathogens. Soil rhizobia also promote nitrogen fixation, which mediates host nutrient levels and defenses. However, whether aboveground herbivores affect the function of soil rhizobia remains poorly understood. We assessed reciprocal interactions between Sitona lineatus, a chewing herbivore, and pea (Pisum sativum) plants grown with or without rhizobia (Rhizobium leguminosarum biovar viciae). We also examined the underlying plant-defense and nutritional mechanisms of these interactions. In our experiments, soil rhizobia influenced feeding and herbivory by chewing herbivores. Leaf defoliation by S. lineatus was lower on plants treated with rhizobia, but these insects had similar amino acid levels compared to those on un-inoculated plants. Plants grown with soil rhizobia had increased expression of gene transcripts associated with phytohormone-mediated defense, which may explain decreased susceptibility to S. lineatus. Rhizobia also induced expression of gene transcripts associated with physical and antioxidant-related defense pathways in P. sativum. Conversely, S. lineatus feeding reduced the number of root nodules and nodule biomass, suggesting a disruption of the symbiosis between plants and rhizobia. Our study shows that aboveground herbivores can engage in mutually antagonistic interactions with soil microbes mediated through a multitude of plant-mediated pathways.

ecology↗

bioRxiv2021

The potyviral protein 6K1 reduces plant protease activity during Turnip mosaic virus infection

O_LIPotyviral genomes encode just 11 major proteins and multifunctionality is associated to most of these proteins at different stages of virus life cycle. The potyviral protein 6K1 is required for potyvirus replication at the early stages of viral infection and may mediate cell-to-cell movement at later stages. C_LIO_LIOur study demonstrates that the 6K1 protein from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and transcripts that encode cysteine protease inhibitors when expressed in trans in Nicotiana benthamiana relative to controls. Furthermore, 6K1 stability increases when lipoxygenase and cysteine protease activity is inhibited chemically, linking a mechanism to the rapid turnover of 6K1 when expressed in trans. C_LIO_LIUsing transient expression, we show 6K1 is degraded rapidly at early time points in the infection process, whereas at later stages of infection protease activity is reduced and 6K1 becomes more stable, resulting in higher TuMV accumulation in systemic leaves. There was no impact of 6K1 transient expression on TuMV accumulation in local leaves. C_LIO_LITogether, these results suggest a novel function for the TuMV 6K1 protein which has not been reported previously and enhances our understanding of the complex interactions occurring between plants and potyviruses. C_LI

plant biology↗

bioRxiv2021

Prefusion conformation of SARS-CoV-2 receptor-binding domain favors interactions with human receptor ACE2

A new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2), poses a serious threat across continents, leading the World Health Organization to declare a Public Health Emergency of International Concern. In order to block the entry of the virus into human host cells, major therapeutic and vaccine design efforts are now targeting the interactions between the SARS-CoV-2 spike (S) glycoprotein and the human cellular membrane receptor angiotensin-converting enzyme, hACE2. By analyzing cryo-EM structures of SARS-CoV-2 and SARS-CoV-1, we report here that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) that binds with hACE2 has expanded in size, undergoing a large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits higher intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bonds in the CoV-2 complex. Further interface analysis has shown that interfacial water promotes and stabilizes the formation of CoV-2/hACE2 complex. This interaction has caused a significant structural rigidification, favoring proteolytic processing of S protein for the fusion of the viral and cellular membrane. Moreover, conformational dynamics simulations of RBD motions in SARS-CoV-2 and SARS-CoV-1 point to the role in modification in the RBD dynamics and their likely impact on infectivity.

bioinformatics↗

bioRxiv2021

Small but complete: liquid-liquid phase separation in minimalistic peptides

Liquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is of urgent importance but remains challenging due to the enormous sequence space and difficulties of existing methodologies in characterizing the phase behavior of ultrashort peptides. Here we demonstrate computational tools to efficiently quantify the microscopic fluidity and density of liquid-condensates/solid-aggregates and the temperature-dependent phase diagram of peptides. Utilizing our approaches, we comprehensively predict the LLPS abilities of all 400 dipeptide combinations of coded amino acids based on 492 micro-second molecular dynamics simulations, and observe the occurrences of spontaneous LLPS. We identify 54 dipeptides that form solid-like aggregates and three categories of dipeptides with high LLPS propensity. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy on four representative dipeptides (WW, QW, GF, and VI). Phase coexistence diagrams are constructed to explore the temperature dependence of LLPS. Our results reveal that aromatic moieties are crucial for a dipeptide to undergo LLPS, and hydrophobic and polar components are indispensable. We demonstrate for the first time that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting that LLPS is a general property of peptides/proteins, independent of their sequence length. This study provides a computational and experimental approach to the prediction and characterization of the phase behavior of minimalistic peptides, and will be helpful for understanding the sequence-dependence and molecular mechanism of protein phase separation. SignificanceProtein liquid-liquid phase separation (LLPS) is associated with human health and diseases. Identifying the minimalistic building blocks and sequence determinants of LLPS is of urgent importance but remains computationally challenging partially due to the lack of methodologies characterizing the liquid condensates. Herein we provide approaches to evaluate LLPS ability of dipeptides, and screen all 400 dipeptides by MD simulations combined with multi-bead-per-residue models which capture key interactions driving LLPS that are missing in one-bead-per-residue models. Three categories of LLPS dipeptides are identified and the experimentally-verified QW dipeptide is by far the smallest LLPS system. Our results suggest that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, and LLPS is a general property of peptides/proteins, independent of their length.

biophysics↗

bioRxiv2021

Plant responses to multiple antagonists are mediated by order of attack and phytohormone crosstalk

Plants are often attacked by multiple antagonists, and traits of the attacking organisms, and their order of arrival onto hosts, may affect plant defenses. However, few studies have assessed how multiple antagonists, and varying attack order, affect plant defense or nutrition. To address this, we assessed defensive and nutritional responses of Pisum sativum plants after attack by a vector herbivore (Acrythosiphon pisum), a non-vector herbivore (Sitona lineatus), and a pathogen (Pea enation mosaic virus, PEMV). We show PEMV-infectious A. pisum induced several pathogen-specific plant defense signals, but these defenses were inhibited when S. lineatus was present in peas infected with PEMV. In contrast, feeding by S. lineatus induced anti-herbivore defense signals, but these defenses were enhanced by PEMV. Sitona lineatus also increased abundance of plant amino acids, but only when they attacked after PEMV-infectious A. pisum. Our results suggest that diverse communities of biotic antagonists alter defense and nutritional traits of plants through complex pathways that depend on the identity of attackers and their order of arrival onto hosts. Moreover, we show interactions among a group of biotic stressors can vary along a spectrum from antagonism to enhancement/synergism based on the identity and order of attackers, and these interactions are mediated by a multitude of phytohormone pathways.

ecology↗

bioRxiv2019

Allosteric regulation of Glutamate dehydrogenase deamination activity

Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of the allosteric network behind GDH regulation, a clear understanding of the enzyme dynamics involved in the process is limited to the activator Adenosine diphosphate (ADP) and the inhibitor Guanosine triphosphate (GTP). Presently, the identification of further factors involved in the allosteric network is paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our structural analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH bound GDH produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.

systems biology↗