DRP1: At the Crossroads of Dysregulated Mitochondrial Dynamics and Altered Cell Signaling in Cancer Cells.

In the past decade, compelling evidence has accumulated that highlights the role of various subcellular structures in human disease conditions. Dysregulation of these structures greatly impacts cellular function and, thereby, disease conditions. One such organelle extensively studied for its role in several human diseases, especially cancer, is the mitochondrion. DRP1 is a GTPase that is considered the master regulator of mitochondrial fission and thereby also affects the proper functioning of the organelle. Altered signaling pathways are a distinguished characteristic of cancer cells. In this review, we aim to summarize our current understanding of the interesting crosstalk between the mitochondrial structure-function maintained by DRP1 and the signaling pathways that are affected in cancer cells. We highlight the structural aspects of DRP1, its regulation by various modifications, and the association of the protein with various cellular pathways altered in cancer. A better understanding of this association may help in identifying potential pharmacological targets for novel therapies in cancer.

Insights into the role of the conserved GTPase domain residues T62 and S277 in yeast Dnm1.

Mitochondrial division is a highly regulated process. The master regulator of this process is the multi-domain, conserved protein called Dnm1 in yeast. In this study, we systematically analyzed two residues, T62 and S277, reported to be putatively phosphorylated in the GTPase domain of the protein. These residues lie in the G2 and G5 motifs of the GTPase domain. Both residues are important for the function of the protein, as evident from in vivo and in vitro analysis of the non-phosphorylatable and phosphomimetic variants. Dnm1T62A/D and Dnm1S277A/D showed differences with respect to the protein localization and puncta dynamics in vivo, albeit both were non-functional as assessed by mitochondrial morphology and GTPase activity. Overall, the secondary structure of the protein variants was unaltered, but local conformational changes were observed. Interestingly, both Dnm1T62A/D and Dnm1S277A/D exhibited dominant-negative behavior when expressed in cells containing endogenous Dnm1. To our knowledge, we report for the first time a single residue (S277) change that does not alter the localization of Dnm1 but makes it non-functional in a dominant-negative manner. Intriguingly, the two residues analyzed in this study are present in the same domain but exhibit variable effects when mutated to alanine or aspartic acid.

Mind the gap: Methods to study membrane contact sites.

Organelles are dynamic entities whose functions are essential for the optimum functioning of cells. It is now known that the juxtaposition of organellar membranes is essential for the exchange of metabolites and their communication. These functional apposition sites are termed membrane contact sites. Dynamic membrane contact sites between various sub-cellular structures such as mitochondria, endoplasmic reticulum, peroxisomes, Golgi apparatus, lysosomes, lipid droplets, plasma membrane, endosomes, etc. have been reported in various model systems. The burgeoning area of research on membrane contact sites has witnessed several manuscripts in recent years that identified the contact sites and components involved. Several methods have been developed to identify, measure and analyze the membrane contact sites. In this manuscript, we aim to discuss important methods developed to date that are used to study membrane contact sites.

Production of Bioactive Human PAX4 Protein from E. coli.

Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for ß-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (PAX4 protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into Escherichia coli (E. coli) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L E. coli culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.

Acyl CoA oxidase: from its expression, structure, folding, and import to its role in human health and disease.

ß-oxidation of fatty acids is an important metabolic pathway and is a shared function between mitochondria and peroxisomes in mammalian cells. On the other hand, peroxisomes are the sole site for the degradation of fatty acids in yeast. The first reaction of this pathway is catalyzed by the enzyme acyl CoA oxidase housed in the matrix of peroxisomes. Studies in various model organisms have reported the conserved function of the protein in fatty acid oxidation. The importance of this enzyme is highlighted by the lethal conditions caused in humans due to its altered function. In this review, we discuss various aspects ranging from gene expression, structure, folding, and import of the protein in both yeast and human cells. Further, we highlight recent findings on the role of the protein in human health and aging, and discuss the identified mutations in the protein associated with debilitating conditions in patients.

Biological activity of recombinant human PDX1 protein produced from Escherichia coli.

Pancreatic and duodenum homeobox 1 (PDX1) is considered as a pivotal transcription factor that acts as a "master regulator" in pancreatogenesis and maintenance of ß-cells. Earlier study has reported that PDX1 also functions as a tumor suppressor in human gastric cancer cells by inhibiting cell growth. Here, we report the bioactivity of the purified human PDX1 fusion protein using various assays like cell migration, proliferation, cell cycle analysis, and gene expression. In cancer cells, recombinant PDX1 protein reduced cell migration and proliferation, and arrested cell growth by inducing apoptosis in gastric cancer cells. In pancreatic ductal cancer cells, the application of the PDX1 protein resulted in the induction of insulin gene expression. The results of these experiments demonstrate the biological activity imparted by recombinant human PDX1 fusion protein on gastric and pancreatic cancer cells and its usefulness as a biological tool to elucidate its function in various cellular processes.

Sporadic SNCA mutations A18T and A29S exhibit variable effects on protein aggregation, cell viability and oxidative stress.

BACKGROUND: α-synuclein aggregation is the hallmark feature of Parkinson's disease. Both familial and sporadic forms of the disease exhibit this feature. Several mutations have been identified in patients and are associated with the disease pathology. METHODS AND RESULTS: We have used site-directed mutagenesis to generate α-synuclein mutant variants tagged with GFP. Fluorescence microscopy, flow cytometry, western blotting, cell viability and oxidative stress analysis were performed to investigate the effect of two less studied α-synuclein variants. In this study we characterized two less studied α-synuclein mutations, A18T and A29S, in the well-established yeast model. Our data shows variable expression, distribution and toxicity of the protein in the mutant variants A18T, A29S, A53T and WT. The cells expressing the double mutant variant A18T/A53T showed the most increase in the aggregation phenotype and also depicted reduced viability suggesting a more substantial effect of this variant. CONCLUSION: The outcome of our study highlights the variable localization, aggregation phenotype and toxicity of the studied α-synuclein variants. This underscores the importance of in-depth analysis of every disease-associated mutation which may result in variable cellular phenotype.

Characterization of the Multiple Domains of Pex30 Involved in Subcellular Localization of the Protein and Regulation of Peroxisome Number.

Pex30 is a peroxisomal protein whose role in peroxisome biogenesis via the endoplasmic reticulum has been established. It is a 58 KDa multi-domain protein that facilitates contact site formation between various organelles. The present study aimed to investigate the role of various domains of the protein in its sub-cellular localization and regulation of peroxisome number. For this, we created six truncations of the protein (1-87, 1-250, 1-352, 88-523, 251-523 and 353-523) and tagged GFP at the C-terminus. Biochemical methods and fluorescence microscopy were used to characterize the effect of truncation on expression and localization of the protein. Quantitative analysis was performed to determine the effect of truncation on peroxisome number in these cells. Expression of the truncated variants in cells lacking PEX30 did not cause any effect on cell growth. Interestingly, variable expression and localization of the truncated variants in both peroxisome-inducing and non-inducing medium was observed. Truncated variants depicted different distribution patterns such as punctate, reticulate and cytosolic fluorescence. Interestingly, lack of the complete dysferlin domain or C-Dysf resulted in increased peroxisome number similar to as reported for cells lacking Pex30. No contribution of this domain in the reticulate distribution of the proteins was also observed. Our results show an interesting role for the various domains of Pex30 in localization and regulation of peroxisome number.

The nexus between peroxisome abundance and chronological ageing in Saccharomyces cerevisiae.

Ageing is characterized by changes in several cellular processes, with dysregulation of peroxisome function being one of them. Interestingly, the most conserved function of peroxisomes, ROS homeostasis, is strongly associated with ageing and age-associated pathologies. Previous studies have identified a role for peroxisomes in the regulation of chronological lifespan in yeast. In this study, we report the effect of altered peroxisome number on the chronological lifespan of yeast in two different growth media conditions. Three mutants, pex11, pex25 and pex27, defective in peroxisome fission, have been thoroughly investigated for the chronological lifespan. Reduced chronological lifespan of all the mutants was observed in peroxisome-inducing growth conditions. Furthermore, the combined deletion pex11pex25 exhibited the most prominent reduction in lifespan. Interestingly altered peroxisomal phenotype upon ageing was observed in all the cells. Increased ROS accumulation and reduced catalase activity was exhibited by chronologically aged mutant cells. Interestingly, mutants with reduced number of peroxisomes concomitantly also exhibited an accumulation of free fatty acids and increased number of lipid droplets. Taken together, our results reveal a previously unrealized effect of fission proteins in the chronological lifespan of yeast.

Peroxisome biogenesis and inter-organelle communication: an indispensable role for Pex11 and Pex30 family proteins in yeast.

Peroxisomes are highly dynamic organelles present in most eukaryotic cells. They also play an important role in human health and the optimum functioning of cells. An extensive repertoire of proteins is associated with the biogenesis and function of these organelles. Two protein families that are involved in regulating peroxisome number in a cell directly or indirectly are Pex11 and Pex30. Interestingly, these proteins are also reported to regulate the contact sites between peroxisomes and other cell organelles such as mitochondria, endoplasmic reticulum and lipid droplets. In this manuscript, we review our current knowledge of the role of these proteins in peroxisome biogenesis in various yeast species. Further, we also discuss in detail the role of these protein families in the regulation of inter-organelle contacts in yeast.

Increased peroxisome proliferation is associated with early yeast replicative ageing.

Peroxisomes are single membrane-bound organelles ubiquitously present in several cell types and are associated with cell and tissue-specific functions. Their role in cellular ageing is under investigation in various model systems. Metabolism of cellular reactive oxygen species is a universal function performed by these organelles. In this study, we investigated alterations in peroxisome number upon early replicative ageing of yeast cells. Increase in the number of peroxisomes in replicatively aged mother cells of wild-type yeast was observed when cultured in both peroxisome-inducing and non-inducing medium. Further, we investigated if this increase in peroxisome number in replicatively aged cells is due to enhanced peroxisome proliferation. For this, the number of peroxisomes in replicatively aged mother cells of pex11, pex25 and pex11pex25 was analysed. Increased percentage of aged cells was observed in pex25 and pex11pex25 cells cultured in peroxisome-inducing oleic acid medium. Interestingly, when cultured in oleic acid, young mother cells devoid of Pex11 showed reduced peroxisome proliferation compared to old mother cells. Induced activity of the antioxidant enzyme catalase and reduced accumulation of reactive oxygen species were reported in all studied strains when cultured in oleic acid medium. Further, our data also suggest that replicatively aged cells with increased peroxisome number also display mitochondrial dysfunction and fragmentation in all the strains studied. In conclusion, our data suggests a correlation between increase in peroxisome number and replicative age of yeast cells and interestingly this increase seems to be partly dependent on the fission proteins.

Pex30 undergoes phosphorylation and regulates peroxisome number in Saccharomyces cerevisiae.

Pex30 is a dysferlin domain-containing protein whose role in peroxisome biogenesis has been studied by several research groups. Notably, recent studies have linked this protein to peroxisomes, endoplasmic reticulum and lipid bodies in Saccharomyces cerevisiae. Phosphoproteome studies of S. cerevisiae have identified several phosphorylation sites in Pex30. In this study we expressed and purified Pex30 from its native host. Analysis of the purified protein by circular dichroism spectroscopy showed that it retained its secondary structure and revealed primarily a helical structure. Further phosphorylation of Pex30 at three residues, Threonine 60, Serine 61 and Serine 511 was identified by mass spectrometry in this study. To understand the importance of this post-translational modification in peroxisome biogenesis, the identified residues were mutated to both non-phosphorylatable (alanine) and phosphomimetic (aspartic acid) variants. Upon analysis of the mutant variants by fluorescence microscopy, no alteration in the localization of the protein to ER and peroxisomes was observed. Interestingly, reduced number of peroxisomes were observed in cells expressing phosphomimetic mutations when cultured in peroxisome-inducing conditions. Our data suggest that phosphorylation and dephosphorylation of Pex30 may promote distinct interactions essential in regulating peroxisome number in a cell.

In vitro, in vivo and in silico rationale for the muscle loss due to therapeutic drugs used in the treatment of Mycobacterium tuberculosis infection.

Tuberculosis globally affects millions of people every year and is responsible for high rates of mortality and morbidity in tropical countries like India. The treatment of tuberculosis involves using the first line of drugs especially Isoniazid, Pyrazinamide, Streptomycin, Ethambutol and Rifampicin for treatment under the DOTS (Directly Observed Treatment Shots) regime which can last up to minimum of six months. These drugs although widely used against Mycobacterium tuberculosis has given rise to multi drug resistant (MDR) tuberculosis strain. It has been observed widely that prolonged drug treatment for tuberculosis patient has rendered several side effects that include increasing muscle wasting and malnutrition. In our study, we have investigated the role of these major tuberculosis drugs namely Rifampicin, Streptomycin, Isoniazid, Pyrazinamide, and Ethambutol on actin polymerization which are famously known to be a central player in the sarcomere region of the muscle in human body. For in vitro studies, we have used biophysical approaches such as 90° scattering assay (RLS), size exclusion chromatography (SEC), Dynamic light scattering (DLS), Circular dichroism spectroscopy (CD), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), kinetic analysis to understand the time taken to break down effect of above mentioned drugs on actin disruption. In vivo analysis was carried out on yeast Δend3 mutants which are rich in F-actin filaments in order to understand the effect of the aforementioned drugs in rendering the muscle wasting phenomenon in tuberculosis. Furthermore, we also carried out in silico analysis to understand the probable modes of binding of these drugs on actin filaments.Communicated by Ramaswamy H. Sarma.

The role of Colchicine on actin polymerization dynamics: as a potent anti-angiogenic factor.

Over the years, cancer research has focused on different strategies to discover drugs and therapies to treat the metastatic stage of cancer. This stage depends upon the type, and the cause of cancer. One of the central facts about any cancer invasion is the formation of new blood vessels that provide nutrients to these uncontrollably dividing cells. This phenomenon is called angiogenesis and is responsible for tumor progression and metastasis. Tumor angiogenesis is a sequential process wherein various angiogenic factors produced by tumor cells bind to receptors of endothelial cells. This stimulates the cytoskeletal protein, especially actin to reorganize themselves and undergo the process of canalization. The driving force for such membrane transformation is spatially and temporally-regulated by polymerization of submembrane actin filaments. So far, Colchicine has been studied for its effectiveness in controlling microtubule reorganization during cell division, but its role is far from understood on actin polymerization. In our current study, we report the effect of Colchicine on actin polymerization dynamics using biophysical analysis like Right light scattering (RLS), Dynamic light scattering (DLS), Circular dichroism (CD) analysis, Scanning electron microscopy (SEM) study. Isothermal titration calorimetry (ITC) and kinetic measurements. Isothermal titration calorimetry (ITC) indicates multiple site binding for colchicine with actin aggregates. We have checked the in vivo effect of colchicine using end3 cells of Saccharomyces cerevisiae. We also report the anti-angiogenesis activity of colchicine via ex-ovo chicken chorioallantoic membrane (CAM) assay. We predict the target site of binding for the drug by docking studies. Based on our findings, we suggest the 'drug-repurposed' function for colchicine as a potential anti-angiogenic candidate.Communicated by Ramaswamy H. Sarma.

Mitochondrial dynamics and its impact on human health and diseases: inside the DRP1 blackbox.

Mitochondria are essential organelles that play a significant role in various cellular processes apart from providing energy in eukaryotic cells. An intricate link between mitochondrial structure and function is now unequivocally accepted. Several molecular players have been identified, which are important in maintaining the structure of the organelle. Dynamin-related protein 1 (DRP1) is one such conserved protein that is a vital regulator of mitochondrial dynamics. Multidisciplinary studies have helped elucidate the structure of the protein and its mechanism of action in great detail. Mutations in various domains of the protein have been identified that are associated with debilitating conditions in patients. The involvement of the protein in disease conditions such as neurodegeneration, cancer, and cardiovascular disorders is also gaining attention. The purpose of this review is to highlight recent findings on the role of DRP1 in human disease conditions and address its importance as a therapeutic target.

Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli.

Neurogenin 3 (NGN3) transcription factor is vital for the development of endocrine cells of the intestine and pancreas. NGN3 is also critical for the neural precursor cell determination in the neuroectoderm. Additionally, it is one of the vital transcription factors for deriving human ß-cells from specialized somatic cells. In the current study, the production and purification of the human NGN3 protein from Escherichia coli (E. coli) is reported. First, the 642 bp protein-coding nucleotide sequence of the NGN3 gene was codon-optimized to enable enhanced protein expression in E. coli strain BL21(DE3). The codon-optimized NGN3 sequence was fused in-frame to three different fusion tags to enable cell penetration, nuclear translocation, and affinity purification. The gene insert with the fusion tags was subsequently cloned into an expression vector (pET28a( +)) for heterologous expression in BL21(DE3) cells. A suitable genetic construct and the ideal expression conditions were subsequently identified that produced a soluble form of the recombinant NGN3 fusion protein. This NGN3 fusion protein was purified to homogeneity (purity > 90%) under native conditions, and its secondary structure was retained post-purification. This purified protein, when applied to human cells, did not induce cytotoxicity. Further, the cellular uptake and nuclear translocation of the NGN3 fusion protein was demonstrated followed by its biological activity in PANC-1 cells. Prospectively, this recombinant protein can be utilized for various biological applications to investigate its functionality in cell reprogramming, biological processes, and diseases.

Post-translational modifications of proteins associated with yeast peroxisome membrane: An essential mode of regulatory mechanism.

Peroxisomes are single membrane-bound organelles important for the optimum functioning of eukaryotic cells. Seminal discoveries in the field of peroxisomes are made using yeast as a model. Several proteins required for the biogenesis and function of peroxisomes are identified to date. As with proteins involved in other major cellular pathways, peroxisomal proteins are also subjected to regulatory post-translational modifications. Identification, characterization and mapping of these modifications to specific amino acid residues on proteins are critical toward understanding their functional significance. Several studies have tried to identify post-translational modifications of peroxisomal proteins and determine their impact on peroxisome structure and function. In this manuscript, we provide an overview of the various post-translational modifications that govern the peroxisome dynamics in yeast.

Contribution of yeast models to virus research.

Time and again, yeast has proven to be a vital model system to understand various crucial basic biology questions. Studies related to viruses are no exception to this. This simple eukaryotic organism is an invaluable model for studying fundamental cellular processes altered in the host cell due to viral infection or expression of viral proteins. Mechanisms of infection of several RNA and relatively few DNA viruses have been studied in yeast to date. Yeast is used for studying several aspects related to the replication of a virus, such as localization of viral proteins, interaction with host proteins, cellular effects on the host, etc. The development of novel techniques based on high-throughput analysis of libraries, availability of toolboxes for genetic manipulation, and a compact genome makes yeast a good choice for such studies. In this review, we provide an overview of the studies that have used yeast as a model system and have advanced our understanding of several important viruses. KEY POINTS: • Yeast, a simple eukaryote, is an important model organism for studies related to viruses. • Several aspects of both DNA and RNA viruses of plants and animals are investigated using the yeast model. • Apart from the insights obtained on virus biology, yeast is also extensively used for antiviral development.

Mimicking human Drp1 disease-causing mutations in yeast Dnm1 reveals altered mitochondrial dynamics.

The dynamin-related protein 1 (Drp1) and its homologs in various eukaryotes are essential to maintain mitochondrial morphology and regulate mitochondrial division. Several mutations in different domains of Drp1 have been reported, which result in debilitating conditions. Four such disease-causing mutations of the middle domain of Drp1 were mimicked in the yeast dynamin-related GTPase (Dnm1) and were characterized in this study. Mitochondrial morphology and protein function were observed to be altered to a variable extent in cells expressing the mutated variants of Dnm1. Several aspects related to the protein such as punctate formation, localization to mitochondria, dynamic behavior and structure were analyzed by microscopy, biochemical studies and molecular dynamics simulations. Significant effects on the protein structure and function were observed in cells expressing A430D and G397D mutations. Overall, our data provide insight into the molecular and cellular alterations resulting from middle domain mutations in Dnm1.

Peroxisomes of the Brain: Distribution, Functions, and Associated Diseases.

Peroxisomes are versatile cell organelles that exhibit a repertoire of organism and cell-type dependent functions. The presence of oxidases and antioxidant enzymes is a characteristic feature of these organelles. The role of peroxisomes in various cell types in human health and disease is under investigation. Defects in the biogenesis of the organelle and its function lead to severe debilitating disorders. In this manuscript, we discuss the distribution and functions of peroxisomes in the nervous system and especially in the brain cells. The important peroxisomal functions in these cells and their role in the pathology of associated disorders such as neurodegeneration are highlighted in recent studies. Although the cause of the pathogenesis of these disorders is still not clearly understood, emerging evidence supports a crucial role of peroxisomes. In this review, we discuss research highlighting the role of peroxisomes in brain development and its function. We also provide an overview of the major findings in recent years that highlight the role of peroxisome dysfunction in various associated diseases.