Biochemistry and Pharmacology - Research Publications
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ItemProtein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli(NATURE PORTFOLIO, 2022-11-28)The correct spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. 'Protein painting methods provide a means to address this gap in knowledge by monitoring the conformational status of proteins within cells at the proteome-wide scale. Here, we demonstrate the ability of a protein painting method employing tetraphenylethene maleimide (TPE-MI) to reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to individual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.
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ItemTrace residue identification, characterization, and longitudinal monitoring of the novel synthetic opioid beta-U10, from discarded drug paraphernalia(WILEY, 2022-05-23)Empirical data regarding dynamic alterations in illicit drug supply markets in response to the COVID-19 pandemic, including the potential for introduction of novel drug substances and/or increased poly-drug combination use at the "street" level, that is, directly proximal to the point of consumption, are currently lacking. Here, a high-throughput strategy employing ambient ionization-mass spectrometry is described for the trace residue identification, characterization, and longitudinal monitoring of illicit drug substances found within >6,600 discarded drug paraphernalia (DDP) samples collected during a pilot study of an early warning system for illicit drug use in Melbourne, Australia from August 2020 to February 2021, while significant COVID-19 lockdown conditions were imposed. The utility of this approach is demonstrated for the de novo identification and structural characterization of β-U10, a previously unreported naphthamide analog within the "U-series" of synthetic opioid drugs, including differentiation from its α-U10 isomer without need for sample preparation or chromatographic separation prior to analysis. Notably, β-U10 was observed with 23 other drug substances, most commonly in temporally distinct clusters with heroin, etizolam, and diphenhydramine, and in a total of 182 different poly-drug combinations. Longitudinal monitoring of the number and weekly "average signal intensity" (ASI) values of identified substances, developed here as a semi-quantitative proxy indicator of changes in availability, relative purity and compositions of street level drug samples, revealed that increases in the number of identifications and ASI for β-U10 and etizolam coincided with a 50% decrease in the number of positive detections and an order of magnitude decrease in the ASI for heroin.
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ItemPre-operative exercise and pyrexia as modifying factors in malignant hyperthermia (MH).(Elsevier BV, 2022-08)Malignant hyperthermia (MH) is a life-threatening reaction triggered by volatile anesthetics and succinylcholine. MH is caused by mutations in the skeletal muscle ryanodine receptor (RYR1) gene, as is rhabdomyolysis triggered by exertion and/or pyrexia. The discrepancy between the prevalence of risk genotypes and actual MH incidence remains unexplained. We investigated the role of pre-operative exercise and pyrexia as potential MH modifying factors. We included cases from 5 MH referral centers with 1) clinical features suggestive of MH, 2) confirmation of MH susceptibility on Contracture Testing (IVCT or CHCT) and/or RYR1 genetic testing, and a history of 3) strenuous exercise within 72 h and/or pyrexia >37.5 °C prior to the triggering anesthetic. Characteristics of MH-triggering agents, surgery and succinylcholine use were collected. We identified 41 cases with general anesthesias resulting in an MH event (GA+MH, n = 41) within 72 h of strenuous exercise and/or pyrexia. We also identified previous general anesthesias without MH events (GA-MH, n = 51) in the index cases and their MH susceptible relatives. Apart from pre-operative exercise and/or pyrexia, trauma and acute abdomen as surgery indications, emergency surgery and succinylcholine use were also more common with GA+MH events. These observations suggest a link between pre-operative exercise, pyrexia and MH.
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ItemA Comparison of Emotional Triggers for Eating in Men and Women with Obesity(MDPI, 2022-10-01)OBJECTIVE: Emotional eating (EE) is prevalent in people seeking obesity treatment and is a contributor to poor weight loss outcomes. We aimed to delineate the emotions most associated with this type of eating, and whether they differ by sex in people undergoing obesity treatment. METHODS: A cross-sectional study recruiting 387 adults from a hospital obesity management service. Emotional eating was measured using the Emotional Eating Scale (EES). Separate analyses included all participants, and those undergoing lifestyle interventions alone or in combination with obesity medication and/or bariatric surgery. RESULTS: A total of 387 people (71% women) participated in the study (n = 187 receiving lifestyle modification alone; n = 200 in combination with additional treatments). Feeling 'bored' was most commonly and most strongly associated with the urge to eat, regardless of sex or treatment. Women had higher scores for total EES, for subscales of depression and anger, and individual feelings of 'blue', 'sad' and 'upset' compared to men. CONCLUSIONS: Understanding why certain emotions differentially trigger an urge to eat in men and women, and finding strategies to break the link between boredom and eating may enable better personalisation of lifestyle interventions for people with obesity.
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ItemNovel Anti-Neuroinflammatory Properties of a Thiosemicarbazone-Pyridylhydrazone Copper(II) Complex(MDPI, 2022-09-01)Neuroinflammation has a major role in several brain disorders including Alzheimer's disease (AD), yet at present there are no effective anti-neuroinflammatory therapeutics available. Copper(II) complexes of bis(thiosemicarbazones) (CuII(gtsm) and CuII(atsm)) have broad therapeutic actions in preclinical models of neurodegeneration, with CuII(atsm) demonstrating beneficial outcomes on neuroinflammatory markers in vitro and in vivo. These findings suggest that copper(II) complexes could be harnessed as a new approach to modulate immune function in neurodegenerative diseases. In this study, we examined the anti-neuroinflammatory action of several low-molecular-weight, charge-neutral and lipophilic copper(II) complexes. Our analysis revealed that one compound, a thiosemicarbazone-pyridylhydrazone copper(II) complex (CuL5), delivered copper into cells in vitro and increased the concentration of copper in the brain in vivo. In a primary murine microglia culture, CuL5 was shown to decrease secretion of pro-inflammatory cytokine macrophage chemoattractant protein 1 (MCP-1) and expression of tumor necrosis factor alpha (Tnf), increase expression of metallothionein (Mt1), and modulate expression of Alzheimer's disease-associated risk genes, Trem2 and Cd33. CuL5 also improved the phagocytic function of microglia in vitro. In 5xFAD model AD mice, treatment with CuL5 led to an improved performance in a spatial working memory test, while, interestingly, increased accumulation of amyloid plaques in treated mice. These findings demonstrate that CuL5 can induce anti-neuroinflammatory effects in vitro and provide selective benefit in vivo. The outcomes provide further support for the development of copper-based compounds to modulate neuroinflammation in brain diseases.
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ItemC-reactive protein, immunothrombosis and venous thromboembolism.(Frontiers Media SA, 2022)C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.
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ItemThe role of mucosal-associated invariant T cells in visceral leishmaniasis(FRONTIERS MEDIA SA, 2022-09-15)Mucosal-associated invariant T (MAIT) cells are restricted by MR1 and are known to protect against bacterial and viral infections. Our understanding of the role of MAIT cells in parasitic infections, such as visceral leishmaniasis (VL) caused by protozoan parasites of Leishmania donovani, is limited. This study showed that in response to L. infantum, human peripheral blood MAIT cells from children with leishmaniasis produced TNF and IFN-γ in an MR1-dependent manner. The overall frequency of MAIT cells was inversely correlated with alanine aminotransferase levels, a specific marker of liver damage strongly associated with severe hepatic involvement in VL. In addition, there was a positive correlation between total protein levels and the frequency of IL-17A+ CD8+ MAIT cells, whereby reduced total protein levels are a marker of liver and kidney damage. Furthermore, the frequencies of IFN-γ+ and IL-10+ MAIT cells were inversely correlated with hemoglobin levels, a marker of severe anemia. In asymptomatic individuals and VL patients after treatment, MAIT cells also produced IL-17A, a cytokine signature associated with resistance to visceral leishmaniasis, suggesting that MAIT cells play important role in protecting against VL. In summary, these results broaden our understanding of MAIT-cell immunity to include protection against parasitic infections, with implications for MAIT-cell-based therapeutics and vaccines. At last, this study paves the way for the investigation of putative MAIT cell antigens that could exist in the context of Leishmania infection.
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ItemBioinformatics Approaches to Predict Mutation Effects in the Binding Site of the Proangiogenic Molecule CD93.(Frontiers Media SA, 2022)The transmembrane glycoprotein CD93 has been identified as a potential new target to inhibit tumor angiogenesis. Recently, Multimerin-2 (MMRN2), a pan-endothelial extracellular matrix protein, has been identified as a ligand for CD93, but the interaction mechanism between these two proteins is yet to be studied. In this article, we aim to investigate the structural and functional effects of induced mutations on the binding domain of CD93 to MMRN2. Starting from experimental data, we assessed how specific mutations in the C-type lectin-like domain (CTLD) affect the binding interaction profile. We described a four-step workflow in order to predict the effects of variations on the inter-residue interaction network at the PPI, based on evolutionary information, complex network metrics, and energetic affinity. We showed that the application of computational approaches, combined with experimental data, allowed us to gain more in-depth molecular insights into the CD93-MMRN2 interaction, offering a platform for developing innovative therapeutics able to target these molecules and block their interaction. This comprehensive molecular insight might prove useful in drug design in cancer therapy.
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ItemEndocytic membrane repair by ESCRT-III controls antigen export to the cytosol during antigen cross-presentation(CELL PRESS, 2022-08-16)Despite its crucial role in initiation of cytotoxic immune responses, the molecular pathways underlying antigen cross-presentation remain incompletely understood. The mechanism of antigen exit from endocytic compartments into the cytosol is a long-standing matter of controversy, confronting two main models: transfer through specific channels/transporters or rupture of endocytic membranes and leakage of luminal content. By monitoring the occurrence of intracellular damage in conventional dendritic cells (cDCs), we show that cross-presenting cDC1s display more frequent endomembrane injuries and increased recruitment of endosomal sorting complex required for transport (ESCRT)-III, the main repair system for intracellular membranes, relative to cDC2s. Silencing of CHMP2a or CHMP4b, two effector subunits of ESCRT-III, enhances cytosolic antigen export and cross-presentation. This phenotype is partially reversed by chemical inhibition of RIPK3, suggesting that endocytic damage is related to basal activation of the necroptosis pathway. Membrane repair therefore proves crucial in containing antigen export to the cytosol and cross-presentation in cDCs.
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ItemMitochondrial microproteins link metabolic cues to respiratory chain biogenesis(CELL PRESS, 2022-08-16)Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.