Bio21 - Research Publications
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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.
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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.
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ItemRespiratory strategy at birth initiates distinct lung injury phenotypes in the preterm lamb lung(BMC, 2022-12-14)BACKGROUND: A lack of clear trial evidence often hampers clinical decision-making during support of the preterm lung at birth. Protein biomarkers have been used to define acute lung injury phenotypes and improve patient selection for specific interventions in adult respiratory distress syndrome. The objective of the study was to use proteomics to provide a deeper biological understanding of acute lung injury phenotypes resulting from different aeration strategies at birth in the preterm lung. METHODS: Changes in protein abundance against an unventilated group (n = 7) were identified via mass spectrometry in a biobank of gravity dependent and non-dependent lung tissue from preterm lambs managed with either a Sustained Inflation (SI, n = 20), Dynamic PEEP (DynPEEP, n = 19) or static PEEP (StatPEEP, n = 11). Ventilation strategy-specific pathways and functions were identified (PANTHER and WebGestalt Tool) and phenotypes defined using integrated analysis of proteome, physiological and clinical datasets (MixOmics package). RESULTS: 2372 proteins were identified. More altered proteins were identified in the non-dependent lung, and in SI group than StatPEEP and DynPEEP. Different inflammation, immune system, apoptosis and cytokine pathway enrichment were identified for each strategy and lung region. Specific integration maps of clinical and physiological outcomes to specific proteins could be generated for each strategy. CONCLUSIONS: Proteomics mapped the molecular events initiating acute lung injury and identified detailed strategy-specific phenotypes. This study demonstrates the potential to characterise preterm lung injury by the direct aetiology and response to lung injury; the first step towards true precision medicine in neonatology.
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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.
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ItemInteracting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down(WILEY, 2022-09)The small G protein Arl5b is localised on the trans-Golgi network (TGN) and regulates endosomes-to-TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane-bound Arl5b(Q70L)-GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP-Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN-localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.
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ItemReaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy(AMER ASSOC ADVANCEMENT SCIENCE, 2022-06-03)Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
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ItemDeep proteomic profiling unveils arylsulfatase A as a non-alcoholic steatohepatitis inducible hepatokine and regulator of glycemic control(NATURE PORTFOLIO, 2022-03-10)Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogate hepatocyte protein secretion in two models of murine NASH to understand how liver-derived factors modulate lipid metabolism and insulin sensitivity in peripheral tissues. We reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism, and identify arylsulfatase A (ARSA) as a hepatokine that is upregulated in NASH and type 2 diabetes. Mechanistically, hepatic ARSA reduces sulfatide content and increases lysophosphatidylcholine (LPC) accumulation within lipid rafts and suppresses LPC secretion from the liver, thereby lowering circulating LPC and lysophosphatidic acid (LPA) levels. Reduced LPA is linked to improvements in skeletal muscle insulin sensitivity and systemic glycemic control. Hepatic silencing of Arsa or inactivation of ARSA's enzymatic activity reverses these effects. Together, this study provides a unique resource describing global changes in hepatokine secretion in NASH, and identifies ARSA as a regulator of liver to muscle communication and as a potential therapeutic target for type 2 diabetes.
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ItemOccupational Allergic Sensitization Among Workers Processing King Crab (Paralithodes camtschaticus) and Edible Crab (Cancer pagurus) in Norway and Identification of Novel Putative Allergenic Proteins(FRONTIERS MEDIA SA, 2021-08-23)Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins. Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis. Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens. Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged.
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ItemMembrane-Enriched Proteomics Link Ribosome Accumulation and Proteome Reprogramming With Cold Acclimation in Barley Root Meristems(FRONTIERS MEDIA SA, 2021-04-30)Due to their sessile nature, plants rely on root systems to mediate many biotic and abiotic cues. To overcome these challenges, the root proteome is shaped to specific responses. Proteome-wide reprogramming events are magnified in meristems due to their active protein production. Using meristems as a test system, here, we study the major rewiring that plants undergo during cold acclimation. We performed tandem mass tag-based bottom-up quantitative proteomics of two consecutive segments of barley seminal root apexes subjected to suboptimal temperatures. After comparing changes in total and ribosomal protein (RP) fraction-enriched contents with shifts in individual protein abundances, we report ribosome accumulation accompanied by an intricate translational reprogramming in the distal apex zone. Reprogramming ranges from increases in ribosome biogenesis to protein folding factors and suggests roles for cold-specific RP paralogs. Ribosome biogenesis is the largest cellular investment; thus, the vast accumulation of ribosomes and specific translation-related proteins during cold acclimation could imply a divergent ribosomal population that would lead to a proteome shift across the root. Consequently, beyond the translational reprogramming, we report a proteome rewiring. First, triggered protein accumulation includes spliceosome activity in the root tip and a ubiquitous upregulation of glutathione production and S-glutathionylation (S-GSH) assemblage machineries in both root zones. Second, triggered protein depletion includes intrinsically enriched proteins in the tip-adjacent zone, which comprise the plant immune system. In summary, ribosome and translation-related protein accumulation happens concomitantly to a proteome reprogramming in barley root meristems during cold acclimation. The cold-accumulated proteome is functionally implicated in feedbacking transcript to protein translation at both ends and could guide cold acclimation.
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ItemPrednisolone Alters Endometrial Decidual Cells and Affects Decidual-Trophoblast Interactions(FRONTIERS MEDIA SA, 2021-04-09)Poor pregnancy outcomes such as recurrent pregnancy loss (RPL) and preeclampsia are associated with impaired decidualization and abnormal trophoblast invasion. Emerging evidence suggests that use of corticosteroids, including prednisolone affects fertility by altering uterine function and may be associated with preeclampsia incidence. In this study, using primary and gestational-age appropriate tissue, we aimed to define the effect of prednisolone on human endometrial stromal fibroblast (hESF) decidualization and determine whether hESF decidualization in the presence of prednisolone would alter hESF regulation of trophoblast function. We found that prednisolone treatment reduced hESF cytokine expression (IL6, IL11, IL18, LIF, and LIFR) but had no effect on hESF expression or secretion of the classic markers of decidualization [prolactin (PRL) and IGFBP1]. Using proteomics we determined that prednisolone altered decidualized hESF protein production, enriching hESF proteins associated with acetylation and mitrochondria. Conditioned media from hESF decidualized in the presence of prednisolone significantly enhanced trophoblast outgrowth and trophoblast mRNA expression of cell motility gene PLCG1 and reduced trophoblast production of PGF. Prednisolone treatment during the menstrual cycle and 1st trimester of pregnancy might alter decidual interactions with other cells, including invasive trophoblast.