Biology subjects

Deshpande, M.

Publications and source records attributed to Deshpande, M..

At least 19 recordsLinked to original sources

bioRxiv2026

An NO-binding Cache domain receptor interacts with a Ser/Thr kinase through a conserved HAMP domain interaction

Cache_heme domains are a family of bacterial heme c proteins that combine a conserved Cache fold with an -helical insertion containing a heme-binding CXXCH motif. We characterize an unusual Cache_heme-HAMP (CHH) receptor from Pseudomonas azotoformans with a periplasmic NO-binding Cache_heme domain, transmembrane region, and a cytoplasmic HAMP domain uncoupled from intrinsic enzymatic output. Genomic analysis reveals that CHH receptors are frequently co-localized with genes encoding Ser/Thr kinases (STPKs) that possess a catalytic domain similar to PknB, but lack peptidoglycan-binding PASTA domains. Using biochemical assays, biolayer interferometry, SEC-MALS, and cryo-electron microscopy, we show that the P. azotoformans CHH receptor binds its operon-associated kinase with nanomolar affinity via conserved C-terminal repeat modules in the kinase composed of two pseudo-symmetric -helical bundles. Cryo-EM structures demonstrate that this interaction orders the otherwise flexible HAMP domain, with the repeated helical domains from the kinase contacting each HAMP subunit symmetrically. Kinase-receptor binding is ATP-independent, but kinase phosphorylation of the receptor at a specific HAMP threonine residue is substantially enhanced when NO binds to the heme c sensor domain. Thus, NO-induced conformational changes that transverse the membrane either modulate substrate accessibility in the HAMP or alter kinase activity directly. These findings potentially define a mode of bacterial NO signaling in which a periplasmic heme c receptor couples ligand sensing to cytoplasmic phosphorylation via a physically associated Ser/Thr kinase, a mechanism distinct from that of cytoplasmic NO sensors. The conserved nature of the kinase recognition motif suggests broader relevance of CHH-STPK interactions across proteobacteria.

biochemistry↗

bioRxiv2026

Evolution of moss leaf-like organs through variations in deeply conserved developmental principles

Leaves and leaf-like organs with laminar structures and determinate growth arose multiple times independently in land plants. The cellular basis of leaf development is well characterized in flowering plants, and molecular studies have shown that the plant hormone auxin plays a central role in this process, orchestrating cellular growth and differentiation. Auxin is also crucial for the formation of phyllids, the leaf-like organs of bryophytes, yet its precise role in morphogenesis remains unclear. More broadly, whether similar developmental principles are shared across distantly related evolutionary lineages is unknown. Here, we combine live-imaging, genetics, pharmacological treatments, and modeling to investigate the cellular and molecular basis of phyllid development in the model moss Physcomitrium patens. By tracking phyllid morphogenesis from a single initial cell to full maturity, we uncover the cellular growth dynamics underlying organ development. We demonstrate that auxin spatially inhibits cell divisions and promotes cellular elongation and differentiation. However, unlike in vascular plants, moss PIN transporters do not participate in polar auxin transport during phyllid development but mainly reduce intracellular auxin concentration. These findings indicate that while auxins role in organogenesis is conserved, its transport mechanisms have diverged across land plants. Overall, our study reveals shared principles of planar organ morphogenesis, highlighting how the repeated deployment of similar developmental strategies, with lineage-specific variations, drove the convergent evolution of leaves and leaf-like organs. One-Sentence SummaryAuxin modulates a conserved developmental program in moss leaf-like organs by spatiotemporally controlling cell division and growth.

plant biology↗

bioRxiv2026

A bacterial NO-binding sensor domain evolved through acquisition of a cytochrome-derived c-type heme-binding motif

Diverse bacterial sensory domains detect intracellular and extracellular cues and relay this information to downstream signaling pathways. Per-Arnt-Sim (PAS) domains are well known to bind cofactors such as heme for intracellular sensing, whereas Cache domains - the largest family of bacterial extracytoplasmic sensor modules - are thought to function exclusively as cofactor-independent ligand-binding domains. Here, we identify and characterize a large family of Cache domains that contain a covalently bound c-type heme. Systematic sequence analysis of the DUF3365/Tll0287 family of unknown function reveals exceptional conservation of a canonical CXXCH heme-binding motif, establishing c-type heme binding as a defining feature of this domain family. Structure- and sequence-based comparisons demonstrate that, despite prior annotation as PAS-like, these domains belong to the Cache superfamily and share a conserved Cache fold augmented by a distinct structural element harboring the heme-binding motif. Heme containing Cache domains are widely distributed across bacterial phyla, with strong enrichment in Gracilicutes, and frequently occur as stand-alone modules, an atypical presentation of Cache domains. Phylogenetic analyses support insertion of a short cytochrome c-derived fragment containing the heme-binding motif into a preexisting Cache scaffold, representing a previously unrecognized mode of sensor-domain evolution. Spectral and ligand-binding analyses indicate that heme containing Cache domains fused to signal-transduction modules have properties typical of nitric oxide, but not oxygen, sensors; whereas stand-alone domains likely have redox-related roles. Our findings expand the functional repertoire of Cache domains to include covalently bound cofactors and reveal fragment-level recruitment of enzymatic motifs as a route for the emergence of bacterial sensory systems.

microbiology↗

bioRxiv2025

An ELISA for discovering protein-protein interaction inhibitors: blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook as a test case

The inhibition of a specific protein-protein interaction is often difficult to achieve in targeted drug design. We report the development and optimization of a general-purpose, readily implemented enzyme-linked immunosorbent assay (ELISA) for high-throughput screening to identify small- molecule inhibitors of protein interactions. This ELISA does not involve the use of any capture antibodies, probes, or compounds coated on the plate and represents a general strategy to identify inhibitors of a given protein-protein interaction. We demonstrate its utility in blocking lysinoalanine crosslinking between subunits of the spirochete flagellar hook by targeting the native form of the FlgE protein, which differs from the strategies used in previous assays. The flagellar hook protein FlgE self-catalyzes the formation of a lysinoalanine (Lal) inter-subunit crosslink that is essential for the motility, and thus, infectivity of spirochetes. Prevention of Lal crosslinking through inhibition with small molecules thus represents an avenue for therapeutic development against spirochete-related diseases, such as Lyme and syphilis. Screening a library of [~]700 compounds with the ELISA confirmed that hexachlorophene, currently the only known inhibitor of Lal crosslinking in FlgE, effectively inhibits the crosslinking reaction. In addition, the assay identified two new potential inhibitors, honokiol and zafirlukast, and several activators which belong to well-known classes of antibiotics.

biochemistry↗

bioRxiv2025

APOBEC3A-Induced DNA Damage Drives Polymerase Theta Dependency and Synthetic Lethality in Cancer

APOBEC3 cytidine deaminases drive cancer evolution. There is an unmet need to target cancer cells with APOBEC3 activity. Here, we identify error-prone theta-mediated end joining (TMEJ) as the main pathway for repairing APOBEC3-induced double-strand breaks (DSBs). Using fluorescent DSB repair reporters and a novel biochemical assay, we demonstrate that APOBEC3A competes with replication protein A (RPA) for single-stranded DNA overhangs, exposing microhomologous sequences to shift DSB repair towards error-prone TMEJ. Genomic analysis of clinical tumor samples confirmed the co-occurrence and proximity between APOBEC3-induced mutational footprints, microhomology-mediated deletions (MMDs), and TMEJ-associated chromosomal instability signatures. Crucially, inhibition of DNA polymerase theta (Pol{theta}) synergizes with APOBEC3A-induced DSBs to induce synthetic lethality in vitro and in vivo. Collectively, our findings identify TMEJ as the preferred mechanism for repairing APOBEC3A-induced DSBs and establish Pol{theta} inhibition as a novel promising strategy to eliminate cancer cells with APOBEC3A activity.

cancer biology↗

bioRxiv2025

Dysregulation of the actin cytoskeleton and FMRP causes polar body protrusion defects in human fragile X premutation and aged oocytes

Women carrying the fragile X premutation (55-200 CGG repeat expansion, PM) are at risk for developing fragile X-associated primary ovarian insufficiency (FXPOI), which is preceded by fragile X-associated diminished ovarian reserve (FXDOR). So far, the cause of FXDOR/FXPOI could not be comprehensively examined due to the scarcity of human ovarian tissue and oocytes. From studies in model systems, it was proposed that molecular abnormalities within the ovaries or a diminished primordial follicle pool cause FXDOR/FXPOI. To elucidate the defects instigating FXDOR/FXPOI, we examined human oocytes obtained from PM carriers undergoing in vitro fertilization (IVF). We found that the number of MII oocytes was reduced suggesting that the maturation of the oocytes is constrained in PM carriers. Furthermore, immature PM oocytes contained abnormal inclusions, irregular ubiquitin levels and DNA breaks. Despite these defects PM oocytes passed the DNA damage checkpoints. However, in anaphase I PM oocytes failed to initiate the protrusion of the first polar body. In addition, these oocytes amassed bundle actin structures, lacked an actin cap and had elevated profilin1 level. Profilin1 limits the formation of branched actin structures which are necessary for actin cap formation and membrane protrusions. Surprisingly, our results suggest that in PM oocytes an increase in FMRP elevates the profilin1 translation, which leads to the cytoskeleton defects and deficiencies in formation of the first polar body. We also analyzed the decline of MII oocytes in aging human ovaries. Similar, we found that the profilin1 expression and formation of the actin cytoskeleton were dysregulated due to appearance of cytoplasmatic FMRP foci in aged human oocytes. Thus, these results reveal that defects during anaphase I hinder the maturation of human oocytes resulting in FXDOR/FXPOI in PM carriers and a reduction in mature oocytes in women with advanced maternal age.

cell biology↗

bioRxiv2024

Inhibitors of lysinoalanine crosslinking in the flagella hook as antimicrobials against spirochetes

Spirochetes are especially invasive bacteria that are responsible for several human diseases, including Lyme disease, periodontal disease, syphilis and leptospirosis. Spirochetes rely on an unusual form of motility based on periplasmic flagella (PFs) to infect hosts and evade the immune system. The flexible hook of these PFs contains a post-translational modification in the form of a lysinoalanine (Lal) crosslink between adjacent subunits of FlgE, which primarily comprise the hook. Lal crosslinking has since been found in key species across phylum and involves residues that are highly conserved. The requirement of the Lal crosslink for motility of the pathogens Treponema denticola (Td) and Borreliella burgdorferi (Bb) establish Lal as a potential therapeutic target for the development of anti-microbials. Herein, we present the design, development and application of a NanoLuc-based high-throughput screen that was used to successfully identify two, structurally related Lal crosslink inhibitors (hexachlorophene and triclosan) from a library of clinically approved small molecules. A structure-activity relationship study further expanded the inhibitor set to a third compound (dichlorophene) and each inhibitor was demonstrated to biochemically block autocatalytic crosslinking of FlgE from several pathogenic spirochetes with varied mechanisms and degrees of specificity. The most potent inhibitor, hexachlorophene, alters Lal crosslinking in cultured cells of Td and reduces bacterial motility in swimming plate assays. Overall, these results provide a proof-of-concept for the discovery and development of Lal-crosslink inhibitors to combat spirochete-derived illnesses.

biochemistry↗

bioRxiv2024

R2DT: a comprehensive platform for visualising RNA secondary structure

RNA secondary (2D) structure visualisation is an essential tool for understanding RNA function. R2DT is a software package designed to visualise RNA 2D structures in consistent, recognisable, and reproducible layouts. The latest release, R2DT 2.0, introduces multiple significant features, including the ability to display position-specific information, such as single nucleotide polymorphisms (SNPs) or SHAPE reactivities. It also offers a new template-free mode allowing visualisation of RNAs without pre-existing templates, alongside a constrained folding mode and support for animated visualisations. Users can interactively modify R2DT diagrams, either manually or using natural language prompts, to generate new templates or create publication-quality images. Additionally, R2DT features faster performance, an expanded template library, and a growing collection of compatible tools and utilities. Already integrated into multiple biological databases, R2DT has evolved into a comprehensive platform for RNA 2D visualisation, accessible at https://r2dt.bio.

bioinformatics↗

bioRxiv2024

Unbiased multiplex antigen screening of Cerebrospinal Fluid detects microbial and autoantigenic epitopes associated with Multiple Sclerosis

To comprehensively investigate the intrathecal antibody profile of multiple sclerosis (MS), we examined the cerebrospinal fluid of 195 patients (92 MS and 103 non-MS) for antibodies using a multiplex unbiased bacteria peptide library. We first tested against Epstein-Barr nuclear antigen 1 (EBNA1) for epitope sites enriched in MS and found a significant enrichment at position 407-419. We then used the data to perform a high-throughput screen against a library of 129 viruses known to infect humans. We discovered several additional epitopes from viruses such as Hantaan virus, Human Herpesvirus 6A and Human respiratory syncytial virus B associated with MS. Besides viral epitopes, we also screened for potential autoantigens of the central nervous system (CNS). We discovered several autoantigenic epitopes in proteins such as ADRB3, HTR3A and MPO that were significantly enriched for MS. Because of previous associations of Toxoplasma gondii infection with MS, we also performed a Toxoplasma gondii specific analysis and discovered additional epitopes enriched for MS. We further assessed epitope-epitope correlations within the patient samples and found distinct patterns of association between these microbial and autoantigenic epitopes. Finally, we performed machine-learning to determine if these epitopes are predictive for MS and found that the model incorporating all the epitopes could most effectively discriminate between MS and non-MS (ROC-AUC score = 0.91). Our results demonstrate the effectiveness of multiplex unbiased screens to detect the identity of potentially cross-reactive antibodies targeting MS CNS epitopes and they can also be used as effective biomarkers for MS. One Sentence SummaryWe performed an unbiased multiplex bacteria peptide antibody library screen on cerebrospinal fluid samples of patients with multiple sclerosis (MS) as well as non-MS controls and detected multiple viral and autoantigenic epitopes that are significantly enriched in MS patient samples.

immunology↗

bioRxiv2024

AAV delivery of GBA1 suppresses alpha-synuclein accumulation in Parkinson's disease models and restores motor dysfunction in a Gaucher's disease model

Biallelic mutations in the glucosylceramidase beta 1 (GBA1) gene are the underlying genetic cause of Gauchers disease (GD), resulting in a deficient lysosomal hydrolase and subsequent accumulation of glycosphingolipids. More recently, GBA1 mutations have been identified as the most prevalent genetic risk factor for Parkinsons disease (PD), associated with more pronounced symptoms characterized by earlier onset and accelerated cognitive decline. In these GBA-associated PD patients the -synuclein pathology is more prominent, and recent data suggest a link between -synucleinopathies and GBA1 mutations. Here, we explored the effect of GBA1 gene supplementation on the GD phenotypes and -synuclein pathology by using the adeno-associated virus (AAV) system. We have compared two AAV serotypes, AAV5 and AAV9, and two different ubiquitous promoters, and demonstrate that both promoters work efficiently albeit not the same in vitro and in vivo. GBA1 overexpression reduces the accumulation of glucosylsphingosine (GlcSph) and restores motor dysfunction in a GD mouse model. We further demonstrate that GBA1 overexpression can dissolve phospho--synuclein aggregation induced by the addition of -synuclein pre-formed fibril (PFF) in a mouse primary neuron model suggesting the direct effect of {beta}-Glucocerebrosidase (GCase) on -synuclein accumulation. In vivo, we show that GCase inhibition can induce insoluble high-molecular-weight -synuclein aggregation and that delivery of GBA1 achieves robust reduction of the -synuclein aggregates in the mouse brain. In summary, GCase expression not only reduces GlcSph, but also restores GD motor dysfunction and removes -synuclein aggregates which are the hallmark for PD and -synucleinopathies. AAV delivery of GBA1 is a powerful approach to restore glucocerebrosidase function and to resolve misfolded -synuclein protein, with applications for GD and PD.

neuroscience↗

bioRxiv2023

Lysinoalanine crosslinking is a conserved post-translational modification in the spirochete flagellar hook

Spirochete bacteria cause Lyme disease, leptospirosis, syphilis and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) catalyzes the formation of covalent lysinoalanine (Lal) crosslinks between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. Although not necessary for hook assembly, Lal is required for motility of Td, presumably due to the stabilizing effect of the crosslink. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal crosslinked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp.. Like with Td, a mutant strain of the Lyme disease pathogen Borreliella burgdorferi unable to form the crosslink has impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveals that the Lal crosslink is a conserved and necessary post-translational modification across the spirochete phylum and may thus represent an effective target for spirochete-specific antimicrobials. Significance StatementThe phylum Spirochaetota contains bacterial pathogens responsible for a variety of diseases, including Lyme disease, syphilis, periodontal disease, and leptospirosis. Motility of these pathogens is a major virulence factor that contributes to infectivity and host colonization. The oral pathogen Treponema denticola produces a post-translational modification (PTM) in the form of a lysinoalanine (Lal) crosslink between neighboring subunits of the flagellar hook protein FlgE. Herein, we demonstrate that representative spirochetes species across the phylum all form Lal in their flagellar hooks. T. denticola and B. burgdorferi cells incapable of forming the crosslink are non-motile, thereby establishing the general role of the Lal PTM in the unusual type of flagellar motility evolved by spirochetes.

microbiology↗

bioRxiv2023

Annotating Macromolecular Complexes in the Protein Data Bank: Improving the FAIRness of Structure Data

Macromolecular complexes are essential functional units in nearly all cellular processes, and their atomic-level understanding is critical for elucidating and modulating molecular mechanisms. The Protein Data Bank (PDB) serves as the global repository for experimentally determined structures of macromolecules. Structural data in the PDB offer valuable insights into the dynamics, conformation, and functional states of biological assemblies. However, the current annotation practices lack standardised naming conventions for assemblies in the PDB, complicating the identification of instances representing the same assembly. In this study, we introduce a method leveraging resources external to PDB, such as the Complex Portal, UniProt and Gene Ontology, to describe assemblies and contextualise them within their biological settings accurately. Employing the proposed approach, we assigned standard names and provided value-added annotations to over 90% of unique assemblies in the PDB. This standardisation of assembly data enhances the PDB, facilitating a deeper understanding of these cellular components. Furthermore, the data standardisation improves the PDBs FAIR attributes, fostering more effective basic and translational research and education across scientific disciplines.

bioinformatics↗

bioRxiv2022

3D-Beacons: Decreasing the gap between protein sequences and structures through a federated network of protein structure data resources

While scientists can often infer the biological function of proteins from their 3-dimensional quaternary structures, the gap between the number of known protein sequences and their experimentally determined structures keeps increasing. A potential solution to this problem is presented by ever more sophisticated computational protein modelling approaches. While often powerful on their own, most methods have strengths and weaknesses. Therefore, it benefits researchers to examine models from various model providers and perform comparative analysis to identify what models can best address their specific use cases. To make data from a large array of model providers more easily accessible to the broader scientific community, we established 3D-Beacons, a collaborative initiative to create a federated network with unified data access mechanisms. The 3D-Beacons Network allows researchers to collate coordinate files and metadata for experimentally determined and theoretical protein models from state-of-the-art and specialist model providers and also from the Protein Data Bank.

bioinformatics↗

bioRxiv2022

PDB ProtVista: A reusable and open-source sequence feature viewer

The PDB ProtVista is a reusable and customisable sequence feature viewer that provides intuitive and detailed 2D visualisation of residue-level annotations while supporting interactive communication with 3D viewers. The Protein Data Bank in Europe (PDBe) team develops and maintains PDB ProtVista. Several public web services use it to display structural and functional annotations such as macromolecular interaction interfaces, intrinsic disorder predictions, sequence variants, and sequence conservation. The PDB ProtVista is freely available from https://github.com/PDBeurope/protvista-pdb. We provide extensive documentation and step-by-step user guides on integrating PDB ProtVista with existing web applications and 3D molecular viewers. We also offer examples of displaying the users custom data and functional annotations for PDB and UniProt entries powered by a rich set of PDBe API endpoints.

bioinformatics↗

bioRxiv2019

Retinal Plasticity: Functional Recovery after Bipolar Cell Loss in the Oxygen Induced Retinopathy Model

The development of non-invasive live ocular imaging and electrophysiological test systems for rodent eyes provides new tools for not only averaged analysis of the entire retina but also the ability to see, test, and compare different subregions of the same retina. These new capabilities provide the possibility for more detailed examinations of local structural and functional relationships within a single eye and the ability to also follow changes longitudinally over time. We have developed protocols based around the Micron-III/IV retinal imaging camera system for combining fluorescent imaging of the neural retinal micro-vasculature by FA (fluorescein angiography), imaging of all neural retinal layers by SD-OCT (Spectral-Domain Ocular Coherence Tomography), and focal "spot" light-targeted electroretinography (Focal-ERG) to relate the local neurovascular unit structure to the inner (photoreceptor) and outer-retinal electrical response to light stimulation. For demonstration purposes we have used the popular mouse oxygen induced retinopathy (OIR) model, which causes radial central patches of retinal neuron loss mostly in zones away from and between the primary retinal arteries and veins. In this model, the loss of central microvasculature is induced developmentally in mouse litters exposed to 75% oxygen from age P7 to P11. Return to room air on P12, causes several days of retinal ischemia during which neurons, mostly of the inner retina, perish. Bipolar and ganglion cell death ends as neovascular growth revascularizes the central retina. This model provides for non-uniform retinal damage as well as gradual progression and resolution over time. The OIR model was used to generate regions of inner retinal neuron loss in B6.Cg-TgThy1-YFP mice. Using image-guided focal-ERG, the dark-adapted mixed rod-cone light response was compared using stimulation of small circular (0.27 mm diameter) target areas located in the central retinas of the same eyes (OIR and control). The same areas of the same retinas were followed over three ages after revascularization (P21, P28 and P42). ConclusionsCombined FA and SD-OCT imaging can provide local geographic specific information on retinal structural changes and be used to select different retinal areas within the same eye for testing of local light response. This analysis strategy can be employed for studies with rodent disease models that do not uniformly impact the entire retinal area. Combining these techniques would also be useful for testing gene and cell replacement therapies in retinal degeneration models where typically a small zone of the retina is treated. Both treated and untreated retinal zones within the eye can be followed non-invasively over many weeks. SUMMARYMouse models utilized for retinal disease research including retinal vascular models can display nonuniform changes over the entire retina. Damage or loss of retinal layers and retinal neurons due to hypoxia can impact some retinal areas while leaving adjacent regions unaltered. Combining vascular imaging by fluoresceine angiography, vascular imaging and retinal layer imaging by SD-OCT, and focal-ERG provides us with new tools to examine retinal structure-function relationships within a single retina.

neuroscience↗

bioRxiv2019

TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein and forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, several neurological disorders display altered dendritic morphology and complexity, which are thought to precede neurodegeneration. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as a new approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescued defects in dendritic branching. Our data therefore provide a novel mechanism by which TDP-43 dysfunction interferes with dendritic branching, and define new pathways for therapeutic intervention in neurodegenerative diseases.

neuroscience↗

bioRxiv2019

The a-isoform of VEGFA165 is a Significantly Stronger Activator of Human Retinal Endothelial Cells compared to the b-isoform

PurposeThere are reports that a b-isoform of Vascular Endothelial Growth Factor-A-165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. While these isoforms appear to have a different ability to activate the VEGF-Receptor-2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signalling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signalling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). MethodsTo determine full dose-response curves for the activation of MAPK (ERK1/2), AKT and VEGFR2, direct in-cell western assays were developed using primary Human Retinal Microvascular Endothelial Cells (HRMECs). Potential differences in dose-response effects on gene expression markers related to endothelial cell / leukocyte adhesion (ICAM1, VCAM1 and SELE) and tight-junctions (CLDN5 and OCLN) were tested by quantitative-PCR. ResultsActivation dose-response analysis revealed much stronger activation of MAPK, AKT and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA165a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA165b. Similar maximum activation of MAPK was achieved by both isoforms at high concentration. Maximum activation of AKT by VEGFA165b was only half of the maximum activation from VEGFA165a. At a lower intermediate dose, where VEGFA165a activated intracellular signalling stronger than VEGFA165b, the changes to VEGFA target gene expression was generally greater with VEGFA165a. ConclusionsIn primary HRMECs, VEGFA165a could maximally activate MAPK and AKT at lower concentrations where VEGFA165b had relatively little effect. The timing for maximal activation of MAPK was similar for both isoforms, which is different than reprorted for non-retinal endothelial cells. While VEGFA165a and VEGFA165b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ablility to activate at least two key intracellular signalling pathways and potentially VEGF target gene expression in primary human retinal endothelial cells.

cell biology↗

bioRxiv2018

Interleukin 22 mediates interleukin 23-induced pathology in newborn mice by disrupting the function of intestinal and pancreatic cells

Mice expressing IL-23 constitutively in the intestine or skin fail to grow and die prematurely. These phenotypes are associated with marked changes in the levels of circulating cytokines and with changes in the transcriptome of the pancreas and intestine. Marked changes are observed in the expression of molecules involved in digestion and absorption of carbohydrates, proteins, and lipids, resulting in a malabsorptive condition. Genetic ablation of IL-22, or one of the subunits of the IL-22R in mice expressing IL-23, restores normal growth and increases the life span of the animals. Mechanistically, IL-22 acts directly at the level of pancreatic acinar cells to decrease expression of the pancreas associated transcription factor 1a (Ptfla), an important transcription factor controlling expression of genes encoding pancreatic enzymes, and acinar cell identity. The results indicate that dysregulated expression of IL-23 and IL-22 has severe consequences in newborns and reveal an unsuspected role for IL-22 in controlling pancreatic enzyme secretion and food absorption.

immunology↗