Characterisation of glyoxalase 1 mutant mouse and glyoxalase 1 copy number alteration

Glyoxalase 1 (Glo1) of the glyoxalase system catalyses the metabolism of the reactive dicarbonyl metabolite, methylglyoxal, and thereby prevents potentially damaging glycation of protein and DNA. Glo1 is hypothesised to be a potential factor in the development of vascular complication of diabetes, such as diabetic nephropathy. The induction of diabetes in mice deficient in Glo1 provides a pre-clinical in vivo model to test this hypothesis. Glo1 mutant mice with putative Glo1 deficiency produced by the International Mouse Knockout Consortium (IMKC) were acquired from the European Mutant Mouse Archive. The initial aim of this study was to study the exacerbation of diabetic nephropathy by Glo1 deficiency in streptozotocin-induced diabetic mice, with an initial objective to confirm Glo1 deficiency in the IMKC Glo1 mutant mouse and subsequent objectives contingent on this. The preliminary studies were unable to confirm Glo1 deficiency in this mouse model and so a revised aim was to characterise the mechanism of compensatory Glo1 expression in the mutant mouse and explore similar occurrence in similar precursor mouse embryonic stem cells (ESCs) and related clinical application. Genotyping of Glo1 mutant mouse offspring by PCR revealed only heterozygotes and wild-type (WT) littermates, and no homozygotes without Glo1 wild-type alleles. Studies of the Glo1 mutant mouse revealed levels of Glo1 activity, protein and mRNA identical to those of wild-type control siblings. Other components of the glyoxalase system were also analysed – activity of glyoxalase 2, concentrations of methylglyoxal (MG) and D-lactate, and tissue protein content and urinary excretion of MG-derived glycation adduct MG-H1 and found no significant change in Glo1 mutant mice, with respect to WT controls. This suggested a functionally normal Glo1 and glyoxalase system in Glo1 mutant mice. Therefore, Glo1 mutant mice have a mutated Glo1 gene but with compensatory Glo1 expression identical to that of WT control. This provided a possible explanation for the unexpected normal phenotype of Glo1 mutant mice reported in the IMKC project. To explore the mechanism of compensatory Glo1 expression, Glo1 copy number was quantified by Taqman® method, normalizing response to transferrin receptor protein-1 (Tfrc). Glo1 mutant mice had 3 copies of Glo1 in all tissues analysed with amplification extending from 3’-end of exon 1 to the 5’-end of exon 6. Taqman copy number assay was established to detect and quantify mutant Glo1Gt(..)Lex and WT alleles. Most mutant mice contained two copies of Glo1 and one mutant copy of Glo1Gt(..)Lex – Glo1(+/+)Gt(..)1Lex. In some cases, however, 2 copies of both Glo1 and mutated Glo1Gt(..)Lex – Glo1(+/+)Gt(..)2Lex were found. Inheritance studies suggested a simple Mendelian inheritance with a WT allele accompanying the Glo1Gt(..)Lex mutant allele on arms of chromosome 17 such that Glo1 deficiency was prevented. This was indeed observed throughout the all breeding of the Glo1 mutant mice. I hypothesised that Glo1 copy number increase may have arisen in the mutant mice during gene trapping by copy number alteration (CNA) induced by increased methylglyoxal concentration, or dicarbonyl stress, in mouse ESCs. To explore and model this, mouse ESCs were cultured with exogenous 200 μM MG under atmospheres containing 20% oxygen - typical of most cell culture conditions, and 3% oxygen - typical of ESCs oxygen exposure in vivo. Incubation of ESCs for 12 days with MG induced CNV increase of Glo1 by up to 16% in both 20% and 3% oxygen atmospheres. Increase in Glo1 CNV at day 12 with MG treatment was associated with an increase in Glo1 protein. Therefore, functional low level CNA of Glo1 was induced by exposure to high levels of exogenous MG. No evidence was found for Glo1 CNA with dicarbonyl stress induced by Glo1 silencing or cell permeable Glo1 inhibitor. Finally, I hypothesised that GLO1 CNA may occur in clinical dicarbonyl stress, a severe example of which is patients with renal failure receiving haemodialysis - associated with ca. 5-fold increase in plasma MG concentration. DNA of peripheral mononuclear cells from healthy subjects and patients with renal failure receiving hemodialysis renal replacement therapy were examined. Human GLO1 copy number was not significantly different between the patients and the control subjects. This requires further investigation in this case and other examples of clinical dicarbonyl stress. From these studies I conclude that the IMKC Glo1 mutant mouse does not exhibit the Glo1 deficiency; rather, it maintains wild-type levels of Glo1 expression through Glo1 copy increase likely induced during gene trapping. Dicarbonyl stress in mouse ESCs in vitro induced low level Glo1 copy number increase – a model of Glo1 CNA in putative gene trapping associated dicarbonyl stress. It is unclear if GLO1 CNA occurs clinically. These findings reveal that focussed copy number alternation of GLO1 may provide a protective response to dicarbonyl stress in some circumstances

Reappraisal of putative glyoxalase 1-deficient mouse and dicarbonyl stress on embryonic stem cells in vitro

Glyoxalase 1 (Glo1) is a cytoplasmic enzyme with a cytoprotective function linked to metabolism of the cytotoxic side product of glycolysis, methylglyoxal (MG). It prevents dicarbonyl stress — the abnormal accumulation of reactive dicarbonyl metabolites, increasing protein and DNA damage. Increased Glo1 expression delays ageing and suppresses carcinogenesis, insulin resistance, cardiovascular disease and vascular complications of diabetes and renal failure. Surprisingly, gene trapping by the International Mouse Knockout Consortium (IMKC) to generate putative Glo1 knockout mice produced a mouse line with the phenotype characterised as normal and healthy. Here, we show that gene trapping mutation was successful, but the presence of Glo1 gene duplication, probably in the embryonic stem cells (ESCs) before gene trapping, maintained wild-type levels of Glo1 expression and activity and sustained the healthy phenotype. In further investigation of the consequences of dicarbonyl stress in ESCs, we found that prolonged exposure of mouse ESCs in culture to high concentrations of MG and/or hypoxia led to low-level increase in Glo1 copy number. In clinical translation, we found a high prevalence of low-level GLO1 copy number increase in renal failure where there is severe dicarbonyl stress. In conclusion, the IMKC Glo1 mutant mouse is not deficient in Glo1 expression through duplication of the Glo1 wild-type allele. Dicarbonyl stress and/or hypoxia induces low-level copy number alternation in ESCs. Similar processes may drive rare GLO1 duplication in health and disease

Improved glycemic control and vascular function in overweight and obese subjects by glyoxalase 1 inducer formulation

Risk of insulin resistance, impaired glycemic control and cardiovascular disease is excessive in overweight and obese populations. We hypothesised that increasing expression of glyoxalase 1 (Glo1) – an enzyme that catalyses the metabolism of reactive metabolite and glycating agent, methylglyoxal – may improve metabolic and vascular health. Dietary bioactive compounds were screened for Glo1 inducer activity in a functional reporter assay, hits confirmed in cell culture and an optimised Glo1 inducer formulation evaluated in a randomised, placebo-controlled crossover clinical trial in 29 overweight and obese subjects. We found trans-resveratrol (tRES) and hesperetin (HESP), at concentrations achieved clinically, synergised to increase Glo1 expression. In highly overweight subjects (BMI >27.5 kg/m2), tRES-HESP co-formulation increased expression and activity of Glo1 (+ 27%. P<0.05), decreased plasma methylglyoxal (-37%, P<0.05) and total body methylglyoxal-protein glycation (-14%, P<0.01). It decreased fasting and postprandial plasma glucose (-5%, P<0.01 and – 6%, P<0.03, respectively), increased Oral Glucose Insulin Sensitivity index (+42 mlmin-1m-2, P<0.02) and improved arterial dilatation ΔFMD/ΔGTND (95%CI 0.13–2.11). In all subjects, it decreased vascular inflammation marker sICAM-1 (-10%, P<0.01). In previous clinical evaluations, tRES and HESP individually were ineffective. tRES-HESP co-formulation could be a suitable treatment for improved metabolic and vascular health in overweight and obese populations

Identification of Phytoconstituents as Potent Inhibitors of Casein Kinase-1 Alpha Using Virtual Screening and Molecular Dynamics Simulations

Casein kinase-1 alpha (CK1&alpha;) is a multifunctional protein kinase that belongs to the serine/threonine kinases of the CK1&alpha; family. It is involved in various signaling pathways associated with chromosome segregation, cell metabolism, cell cycle progression, apoptosis, autophagy, etc. It has been known to involve in the progression of many diseases, including cancer, neurodegeneration, obesity, and behavioral disorders. The elevated expression of CK1&alpha; in diseased conditions facilitates its selective targeting for therapeutic management. Here, we have performed virtual screening of phytoconstituents from the IMPPAT database seeking potential inhibitors of CK1&alpha;. First, a cluster of compounds was retrieved based on physicochemical parameters following Lipinski&rsquo;s rules and PAINS filter. Further, high-affinity hits against CK1&alpha; were obtained based on their binding affinity score. Furthermore, the ADMET, PAINS, and PASS evaluation was carried out to select more potent hits. Finally, following the interaction analysis, we elucidated three phytoconstituents, Semiglabrinol, Curcusone_A, and Liriodenine, posturing considerable affinity and specificity towards the CK1&alpha; binding pocket. The result was further evaluated by molecular dynamics (MD) simulations, dynamical cross-correlation matrix (DCCM), and principal components analysis (PCA), which revealed that binding of the selected compounds, especially Semiglabrinol, stabilizes CK1&alpha; and leads to fewer conformational fluctuations. The MM-PBSA analysis suggested an appreciable binding affinity of all three compounds toward CK1&alpha;

Glyoxalase 1 copy number variation in patients with well differentiated gastroentero-pancreatic neuroendocrine tumours (GEP-NET)

Background: The glyoxalase-1 gene (GLO1) is a hotspot for copy-number variation (CNV) in human genomes. Increased GLO1 copy-number is associated with multidrug resistance in tumour chemotherapy, but prevalence of GLO1 CNV in gastro-entero-pancreatic neuroendocrine tumours (GEP-NET) is unknown. Methods: GLO1 copy-number variation was measured in 39 patients with GEP-NET (midgut NET, n = 25; pancreatic NET, n = 14) after curative or debulking surgical treatment. Primary tumour tissue, surrounding healthy tissue and, where applicable, additional metastatic tumour tissue were analysed, using real time qPCR. Progression and survival following surgical treatment were monitored over 4.2 ± 0.5 years. Results: In the pooled GEP-NET cohort, GLO1 copy-number in healthy tissue was 2.0 in all samples but significantly increased in primary tumour tissue in 43% of patients with pancreatic NET and in 72% of patients with midgut NET, mainly driven by significantly higher GLO1 copy-number in midgut NET. In tissue from additional metastases resection (18 midgut NET and one pancreatic NET), GLO1 copy number was also increased, compared with healthy tissue; but was not significantly different compared with primary tumour tissue. During mean 3 - 5 years follow-up, 8 patients died and 16 patients showed radiological progression. In midgut NET, a high GLO1 copy-number was associated with earlier progression. In NETs with increased GLO1 copy number, there was increased Glo1 protein expression compared to non-malignant tissue. Conclusions: GLO1 copy-number was increased in a large percentage of patients with GEP-NET and correlated positively with increased Glo1 protein in tumour tissue. Analysis of GLO1 copy-number variation particularly in patients with midgut NET could be a novel prognostic marker for tumour progression

MAP/Microtubule Affinity Regulating Kinase 4 Inhibitory Potential of Irisin: A New Therapeutic Strategy to Combat Cancer and Alzheimer’s Disease

Irisin is a clinically significant protein playing a valuable role in regulating various diseases. Irisin attenuates synaptic and memory dysfunction, highlighting its importance in Alzheimer’s disease. On the other hand, Microtubule Affinity Regulating Kinase 4 (MARK4) is associated with various cancer types, uncontrolled neuronal migrations, and disrupted microtubule dynamics. In addition, MARK4 has been explored as a potential drug target for cancer and Alzheimer’s disease therapy. Here, we studied the binding and subsequent inhibition of MARK4 by irisin. Irisin binds to MARK4 with an admirable affinity (K = 0.8 × 107 M−1), subsequently inhibiting its activity (IC50 = 2.71 µm). In vitro studies were further validated by docking and simulations. Molecular docking revealed several hydrogen bonds between irisin and MARK4, including critical residues, Lys38, Val40, and Ser134. Furthermore, the molecular dynamic simulation showed that the binding of irisin resulted in enhanced stability of MARK4. This study provides a rationale to use irisin as a therapeutic agent to treat MARK4-associated diseases

Comparative analysis of web-based programs for single amino acid substitutions in proteins

Single amino-acid substitution in a protein affects its structure and function. These changes are the primary reasons for the advent of many complex diseases. Analyzing single point mutations in a protein is crucial to see their impact and to understand the disease mechanism. This has given many biophysical resources, including databases and web-based tools to explore the effects of mutations on the structure and function of human proteins. For a given mutation, each tool provides a score-based outcomes which indicates deleterious probability. In recent years, developments in existing programs and the introduction of new prediction algorithms have transformed the state-of-the-art protein mutation analysis. In this study, we have performed a systematic study of the most commonly used mutational analysis programs (10 sequence-based and 5 structure-based) to compare their prediction efficiency. We have carried out extensive mutational analyses using these tools for previously known pathogenic single point mutations of five different proteins. These analyses suggested that sequence-based tools, PolyPhen2, PROVEAN, and PMut, and structure-based web tool, mCSM have a better prediction accuracy. This study indicates that the employment of more than one program based on different approaches should significantly improve the prediction power of the available methods

Impact of single amino acid substitutions in Parkinsonism-associated deglycase-PARK7 and their association with Parkinson’s disease

Parkinsonism-associated deglycase-PARK7/DJ-1 (PARK7) is a multifunctional protein having significant roles in inflammatory and immune disorders and cell protection against oxidative stress. Mutations in PARK7 may result in the onset and progression of a few neurodegenerative disorders such as Parkinson’s disease. This study has analyzed the non-synonymous single nucleotide polymorphisms (nsSNPs) resulting in single amino acid substitutions in PARK7 to explore its diseasecausing variants and their structural dysfunctions. Initially, we retrieved the mutational dataset of PARK7 from the Ensembl database and performed detailed analyses using sequence-based and structure-based approaches. The pathogenicity of the PARK7 was then performed to distinguish the destabilizing/deleterious variants. Aggregation propensity, noncovalent interactions, packing density, and solvent accessible surface area analyses were carried out on the selected pathogenic mutations. The SODA study suggested that mutations in PARK7 result in aggregation, inducing disordered helix and altering the strand propensity. The effect of mutations alters the number of hydrogen bonds and hydrophobic interactions in PARK7, as calculated from the Arpeggio server. The study indicated that the alteration in the hydrophobic contacts and frustration of the protein could alter the stability of the missense variants of the PARK7, which might result in disease progression. This study provides a detailed understanding of the destabilizing effects of single amino acid substitutions in PARK7

Identifying promising GSK3β inhibitors for cancer management: a computational pipeline combining virtual screening and molecular dynamics simulations

Glycogen synthase kinase-3 (GSK3β), a serine/threonine protein kinase, has been discovered as a novel target for anticancer drugs. Although GSK3β is involved in multiple pathways linked to the etiology of various cancers, no specific GSK3β inhibitor has been authorized for cancer therapy. Most of its inhibitors have toxicity effects therefore, there is a need to develop safe and more potent inhibitors. In this study, a library of 4,222 anti-cancer compounds underwent rigorous computational screening to identify potential candidates for targeting the binding pocket of GSK3β. The screening process involved various stages, including docking-based virtual screening, physicochemical and ADMET analysis, and molecular dynamics simulations. Ultimately, two hit compounds, BMS-754807 and GSK429286A, were identified as having high binding affinities to GSK3β. BMS-754807 and GSK429286A exhibited binding affinities of −11.9, and −9.8 kcal/mol, respectively, which were greater than that of the positive control (−7.6 kcal/mol). Further, molecular dynamics simulations for 100 ns were employed to optimize the interaction between the compounds and GSK3β, and the simulations demonstrated that the interaction was stable and consistent throughout the study. These hits were also anticipated to have good drug-like properties. Finally, this study suggests that BMS-754807 and GSK429286A may undergo experimental validation to evaluate their potential as cancer treatments in clinical settings

Identification of Phytoconstituents as Potent Inhibitors of Casein Kinase-1 Alpha Using Virtual Screening and Molecular Dynamics Simulations

Casein kinase-1 alpha (CK1&alpha;) is a multifunctional protein kinase that belongs to the serine/threonine kinases of the CK1&alpha; family. It is involved in various signaling pathways associated with chromosome segregation, cell metabolism, cell cycle progression, apoptosis, autophagy, etc. It has been known to involve in the progression of many diseases, including cancer, neurodegeneration, obesity, and behavioral disorders. The elevated expression of CK1&alpha; in diseased conditions facilitates its selective targeting for therapeutic management. Here, we have performed virtual screening of phytoconstituents from the IMPPAT database seeking potential inhibitors of CK1&alpha;. First, a cluster of compounds was retrieved based on physicochemical parameters following Lipinski&rsquo;s rules and PAINS filter. Further, high-affinity hits against CK1&alpha; were obtained based on their binding affinity score. Furthermore, the ADMET, PAINS, and PASS evaluation was carried out to select more potent hits. Finally, following the interaction analysis, we elucidated three phytoconstituents, Semiglabrinol, Curcusone_A, and Liriodenine, posturing considerable affinity and specificity towards the CK1&alpha; binding pocket. The result was further evaluated by molecular dynamics (MD) simulations, dynamical cross-correlation matrix (DCCM), and principal components analysis (PCA), which revealed that binding of the selected compounds, especially Semiglabrinol, stabilizes CK1&alpha; and leads to fewer conformational fluctuations. The MM-PBSA analysis suggested an appreciable binding affinity of all three compounds toward CK1&alpha;