Biology subjects

Cain, K.

Publications and source records attributed to Cain, K..

5 recordsLinked to original sources

bioRxiv2024

Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli

The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

biochemistry↗

bioRxiv2023

The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors

Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent third-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in [~]5-15% of patients. However, it is not fully understand how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.

pharmacology and toxicology↗

bioRxiv2023

Metabolic reprogramming provides a novel approach to overcome resistance to BH3-mimetics in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure and highly resistant to chemotherapy, potentially due to upregulated expression of the pro-survival proteins, BCL2/BCL-XL/MCL-1. Using clinically-relevant models of MPM we show that patient-derived primary MPM cell lines and ex-vivo 3D tumour explants are highly resistant to apoptosis induced by the BCL2/BCL-XL inhibitor, ABT-737. Importantly, we discover that 2-deoxyglucose (2DG), a glycolytic inhibitor, can sensitize MPM cells to ABT-737 and show this correlates with loss of the pro-survival protein, MCL-1. siRNA knockdown of MCL-1 (MCL-1 KD) combined with ABT-737 induced BAX/BAK-dependent, but BIM/PUMA-independent apoptosis, mimicking 2DG/ABT-737 treatment. MCL-1 KD/ABT-737 induced mitochondrial cytochrome c release and caspase-independent inhibition of mitochondrial respiration. Moreover, we observed a hitherto unreported caspase-dependent cleavage of glycolytic enzymes and subsequent inhibition of glycolysis. 2DG inhibited ERK/STAT3 activity, decreased MCL-1 mRNA and protein levels, with concurrent activation of AKT, which limited loss of MCL-1 protein. However, co-treatment with a specific AKT inhibitor, AZD5363, and 2DG/ABT-737 potently induced cell death and inhibited clonogenic cell survival, while in MPM 3D tumour explants MCL-1 protein expression decreased significantly following 2DG or 2DG/AZD5363 treatment. Notably, a similar synergy was observed in MPM cell lines and MPM 3D tumour explants using ABT-737 in combination with the recently developed MCL-1 inhibitor, S63845. Importantly, our study provides a mechanistic explanation for the chemoresistance of MPM and highlights how this can be overcome by a combination of metabolic reprogramming and/or simultaneous targeting of MCL-1 and BCL-2/BCL-XL using BH3-mimetics.

cell biology↗

bioRxiv2021

Characterisation of FADD interactome reveals novel insights into FADD recruitment and signalling at the Death Inducing Signalling Complex (DISC)

Fas-associated death domain protein (FADD) plays a vital role in the extrinsic apoptotic pathway, where it forms an essential component of the death-inducing signaling complex (DISC). However, the precise early molecular events that facilitate recruitment of FADD to the DISC remain poorly defined. Using affinity purification and mass spectrometry we investigated the FADD interactome in untreated cells and following death receptor stimulation to identify novel FADD-interacting proteins. As expected, in death receptor-stimulated samples our analysis identified key components of the DISC such as Caspase-8. In addition, we identified novel binding partners including Transferrin Receptor 1 (TfR1) and Myosin Light Chain Kinase 2 (MYLK2) that are able to modulate FADD recruitment to the DISC and consequently downstream apoptotic signaling. TfR1 is pre-associated with FADD and recruited into the DISC; moreover, our data reveal that TfR1 is also pre-associated with the death receptors, TRAIL-R1 and TRAIL-R2, thereby functioning as a key regulator of DISC formation. In the case of MYLK2, specific binding of FADD to MYLK2 in non-apoptotic cells sequesters FADD from other DISC components ensuring aberrant apoptosis is not initiated. Furthermore, MYLK2 enzymatic activity is required to for it to translocate, in complex with FADD, to sites of DISC-mediated death receptor oligimerization. Taken together, our study highlights the important role that additional novel FADD binding partners play in the regulation of death receptor-mediated apoptotic cell death, in part by modulating FADD recruitment to the DISC.

biochemistry↗

bioRxiv2021

Pharmacologic Therapy for Engraftment Arrhythmia Induced by Transplantation of Human Cardiomyocytes

BackgroundEngraftment arrhythmias (EAs) are observed in large animal studies of intramyocardial transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) for myocardial infarction. Although transient, the risk posed by EA presents a barrier to clinical translation. ObjectivesWe hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. MethodshPSC-CM were transplanted into the infarcted porcine heart by surgical or percutaneous delivery to induce EA. Following a screen of antiarrhythmic agents, a prospective study was conducted to determine the effectiveness of amiodarone plus ivabradine in preventing cardiac death and suppressing EA. ResultsEA was observed in all subjects, and amiodarone-ivabradine treatment was well-tolerated. None of the treated subjects experienced the primary endpoint of cardiac death, unstable EA or heart failure compared to 5/8 (62.5%) in the control cohort (hazard ratio 0.00; 95% confidence interval, 0-0.297; p = 0.002). Overall survival including two deaths in the treated cohort from immunosuppression-related infection showed borderline improvement with treatment (hazard ratio 0.21; 95% confidence interval, 0.03-1.01; p = 0.05). Without treatment, peak heart rate averaged 305 {+/-} 29 beats per min (bpm), whereas in treated subjects peak daily heart rate was significantly restricted to 185{+/-}9 bpm (p = 0.006). Similarly, treatment reduced peak daily EA burden from 96.8 {+/-} 2.9% to 76.5 {+/-} 7.9% (p = 0.003). Antiarrhythmic treatment was safely discontinued after approximately one-month of treatment without recrudescence of arrhythmia. ConclusionsThe risk of engraftment arrhythmia following hPSC-CM transplantation can be reduced significantly by combined amiodarone and ivabradine drug therapy. Condensed AbstractEngraftment arrhythmia (EA) is a transient but serious complication of cardiac remuscularization therapy. Using a porcine model of cardiac remuscularization and EA, ivabradine and amiodarone were independently effective in suppressing tachycardia and arrhythmia, respectively. Baseline amiodarone combined with adjunctive ivabradine successfully prevented cardiac death, unstable EA and heart failure (hazard ratio 0.00; 95% confidence interval, 0-0.297; p = 0.002) and significantly suppressed peak daily heart rate and arrhythmia burden (p=0.006 and 0.003, respectively). Antiarrhythmic treatment was successfully discontinued after one-month without recrudescence of arrhythmia. We conclude that EA can be suppressed by combined amiodarone and ivabradine drug therapy.

pharmacology and toxicology↗