Bio21 - Research Publications
Permanent URI for this collection
Search Results
1 - 10 of 19
-
ItemPhosphoproteomic dysregulation in Huntington's disease mice is rescued by environmental enrichment(OXFORD UNIV PRESS, 2022-11-02)Huntington's disease is a fatal autosomal-dominant neurodegenerative disorder, characterized by neuronal cell dysfunction and loss, primarily in the striatum, cortex and hippocampus, causing motor, cognitive and psychiatric impairments. Unfortunately, no treatments are yet available to modify the progression of the disease. Recent evidence from Huntington's disease mouse models suggests that protein phosphorylation (catalysed by kinases and hydrolysed by phosphatases) might be dysregulated, making this major post-translational modification a potential area of interest to find novel therapeutic targets. Furthermore, environmental enrichment, used to model an active lifestyle in preclinical models, has been shown to alleviate Huntington's disease-related motor and cognitive symptoms. However, the molecular mechanisms leading to these therapeutic effects are still largely unknown. In this study, we applied a phosphoproteomics approach combined with proteomic analyses on brain samples from pre-motor symptomatic R6/1 Huntington's disease male mice and their wild-type littermates, after being housed either in environmental enrichment conditions, or in standard housing conditions from 4 to 8 weeks of age (n = 6 per group). We hypothesized that protein phosphorylation dysregulations occur prior to motor onset in this mouse model, in two highly affected brain regions, the striatum and hippocampus. Furthermore, we hypothesized that these phosphoproteome alterations are rescued by environmental enrichment. When comparing 8-week-old Huntington's disease mice and wild-type mice in standard housing conditions, our analysis revealed 229 differentially phosphorylated peptides in the striatum, compared with only 15 differentially phosphorylated peptides in the hippocampus (statistical thresholds fold discovery rate 0.05, fold change 1.5). At the same disease stage, minor differences were found in protein levels, with 24 and 22 proteins dysregulated in the striatum and hippocampus, respectively. Notably, we found no differences in striatal protein phosphorylation and protein expression when comparing Huntington's disease mice and their wild-type littermates in environmentally enriched conditions. In the hippocampus, only four peptides were differentially phosphorylated between the two genotypes under environmentally enriched conditions, and 22 proteins were differentially expressed. Together, our data indicates that protein phosphorylation dysregulations occur in the striatum of Huntington's disease mice, prior to motor symptoms, and that the kinases and phosphatases leading to these changes in protein phosphorylation might be viable drug targets to consider for this disorder. Furthermore, we show that an early environmental intervention was able to rescue the changes observed in protein expression and phosphorylation in the striatum of Huntington's disease mice and might underlie the beneficial effects of environmental enrichment, thus identifying novel therapeutic targets.
-
ItemProteomic analysis of Sarcoptes scabiei reveals that proteins differentially expressed between eggs and female adult stages are involved predominantly in genetic information processing, metabolism and/or host-parasite interactions(PUBLIC LIBRARY SCIENCE, 2022-12)Presently, there is a dearth of proteomic data for parasitic mites and their relationship with the host animals. Here, using a high throughput LC-MS/MS-based approach, we undertook the first comprehensive, large-scale proteomic investigation of egg and adult female stages of the scabies mite, Sarcoptes scabiei-one of the most important parasitic mites of humans and other animals worldwide. In total, 1,761 S. scabiei proteins were identified and quantified with high confidence. Bioinformatic analyses revealed differentially expressed proteins to be involved predominantly in biological pathways or processes including genetic information processing, energy (oxidative phosphorylation), nucleotide, amino acid, carbohydrate and/or lipid metabolism, and some adaptive processes. Selected, constitutively and highly expressed proteins, such as peptidases, scabies mite inactivated protease paralogues (SMIPPs) and muscle proteins (myosin and troponin), are proposed to be involved in key biological processes within S. scabiei, host-parasite interactions and/or the pathogenesis of scabies. These proteomic data will enable future molecular, biochemical and physiological investigations of early developmental stages of S. scabiei and the discovery of novel interventions, targeting the egg stage, given its non-susceptibility to acaricides currently approved for the treatment of scabies in humans.
-
ItemNo Preview AvailableInhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses(CELL PRESS, 2022-10-10)There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
-
ItemEprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction(AMER ASSOC ADVANCEMENT SCIENCE, 2022-09-16)The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.
-
ItemThermal proteome profiling reveals Haemonchus orphan protein HCO_011565 as a target of the nematocidal small molecule UMW-868(FRONTIERS MEDIA SA, 2022-10-14)Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber's pole worm, and in other socioeconomically important parasitic nematodes. The "hit-to-target" workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.
-
ItemInfluence of protein corona on the interaction of glycogen-siRNA constructs with ex vivo human blood immune cells(ELSEVIER, 2022-09)Glycogen-nucleic acid constructs i.e., glycoplexes are emerging promising platforms for the alteration of gene expression and transcription. Understanding the interaction of glycoplexes with human blood components, such as serum proteins and peripheral blood mononuclear cells (PBMCs), is important to overcome immune cell activation and control biodistribution upon administration of the glycoplexes in vivo. Herein, we investigated the interactions of polyethylene glycol (PEG)ylated and non-PEGylated glycoplexes carrying siRNA molecules with PBMCs isolated from the blood of healthy donors. We found that both types of glycoplexes were non-toxic and were primarily phagocytosed by monocytes without triggering a pro-inflammatory interleukin 6 cytokine production. Furthermore, we investigated the role of the protein corona on controlling the internalization efficiency in immune cells - we found that the adsorption of serum proteins, in particular haptoglobin, alpha-1-antitrypsin and apolipoprotein A-II, onto the non-PEGylated glycoplexes, significantly reduced the uptake of the glycoplexes by PBMCs. Moreover, the non-PEGylated glycoplexes were efficient in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) knockdown in monocytic THP-1 cell line. This study provides an insight into the rational design of glycogen-based nanocarriers for the safe delivery of siRNA without eliciting unwanted immune cell activation and efficient siRNA activity upon its delivery.
-
ItemProtein precoating modulates biomolecular coronas and nanocapsule-immune cell interactions in human blood(ROYAL SOC CHEMISTRY, 2022-09-28)The biomolecular corona that forms on particles upon contact with blood plays a key role in the fate and utility of nanomedicines. Recent studies have shown that precoating nanoparticles with serum proteins can improve the biocompatibility and stealth properties of nanoparticles. However, it is not fully clear how precoating influences biomolecular corona formation and downstream biological responses. Herein, we systematically examine three precoating strategies by coating bovine serum albumin (single protein), fetal bovine serum (FBS, mixed proteins without immunoglobulins), or bovine serum (mixed proteins) on three nanoparticle systems, namely supramolecular template nanoparticles, metal-phenolic network (MPN)-coated template (core-shell) nanoparticles, and MPN nanocapsules (obtained after template removal). The effect of protein precoating on biomolecular corona compositions and particle-immune cell interactions in human blood was characterized. In the absence of a pre-coating, the MPN nanocapsules displayed lower leukocyte association, which correlated to the lower amount (by 2-3 fold) of adsorbed proteins and substantially fewer immunoglobulins (more than 100 times) in the biomolecular corona relative to the template and core-shell nanoparticles. Among the three coating strategies, FBS precoating demonstrated the most significant reduction in leukocyte association (up to 97% of all three nanoparticles). A correlation analysis highlights that immunoglobulins and apolipoproteins may regulate leukocyte recognition. This study demonstrates the impact of different precoating strategies on nanoparticle-immune cell association and the role of immunoglobulins in bio-nano interactions.
-
ItemHidden information on protein function in censuses of proteome foldedness(NATURE PORTFOLIO, 2022-04-14)Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings.
-
ItemA novel PAK4 inhibitor suppresses pancreatic cancer growth and enhances the inhibitory effect of gemcitabine(ELSEVIER SCIENCE INC, 2022-02)Over 95% of Pancreatic ductal adenocarcinomas (PDA) carry mutations in the oncogene KRas which has been proven to be a difficult drug target. P21-activated kinase 4 (PAK4), acts downstream of KRas, and is overexpressed in PDA contributing to its growth and chemoresistance, and thus becomes an attractive therapeutic target. We have developed a new PAK4 inhibitor, PAKib and tested its effect on pancreatic cancer (PC) cell growth in vitro and in a syngeneic mouse model of PC. PAKib suppressed PC cell growth by inducing cell death and cycle arrest. PAKib inhibited PC growth and enhanced the inhibition by gemcitabine of PC in cell culture and in PC mouse model. PAKib acted through multiple signaling pathways involved in cell cycle checkpoints, apoptosis, cell junction, and focal adhesion. These proof-of-concept studies demonstrated the anti-cancer effect of PAKib alone and in combination with gemcitabine and warrant a further clinical investigation.
-
ItemHIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV(American Society for Biochemistry and Molecular Biology, 2020-07-01)Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.