Synergistic Amyloid Switch Triggered by Early Heterotypic Oligomerization of Intrinsically Disordered α-Synuclein and Tau

Amyloidogenic intrinsically disordered proteins, α-synuclein and tau are linked to Parkinson's disease and Alzheimer's disease, respectively. A body of evidence suggests that α-synuclein and tau, both present in the presynaptic nerve terminals, co-aggregate in many neurological ailments. The molecular mechanism of α-synuclein-tau hetero-assembly is poorly understood. Here we show that amyloid formation is synergistically facilitated by heterotypic association mediated by binding-induced misfolding of both α-synuclein and tau K18. We demonstrate that the intermolecular association is largely driven by the electrostatic interaction between the negatively charged C-terminal segment of α-synuclein and the positively charged tau K18 fragment. This heterotypic association results in rapid formation of oligomers that readily mature into hetero-fibrils with a much shorter lag phase compared to the individual proteins. These findings suggested that the critical intermolecular interaction between α-synuclein and tau can promote facile amyloid formation that can potentially lead to efficient sequestration of otherwise long-lived lethal oligomeric intermediates into innocuous fibrils. We next show that a well-known familial Parkinson's disease mutant (A30P) that is known to aggregate slowly via accumulation of highly toxic oligomeric species during the long lag phase converts into amyloid fibrils significantly faster in the presence of tau K18. The early intermolecular interaction profoundly accelerates the fibrillation rate of A30P α-synuclein and impels the disease mutant to behave similar to wild-type α-synuclein in the presence of tau. Our findings suggest a mechanistic underpinning of bypassing toxicity and suggest a general strategy by which detrimental amyloidogenic precursors are efficiently sequestered into more benign amyloid fibrils.     

Effect of Cadmium on Activities of Some Enzymes of Glycolysis and Pentose Phosphate Pathway in Pea

Activities of alcohol dehydrogenase, hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were significantly inhibited by cadmium in germinating pea (Pisum sativum L. cv. Bonneville) seeds. The effect was concentration dependent in the range of 0.25 to 1.0 mM CdCl₂. The magnitude of detrimental effect on these enzymes was reduced during later stage of germination (9 d) largely because of fall in the activities of these enzymes in the control seeds germinated in water. In vitro, activities of hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were inhibited at 0.5 mM Cd²⁺ in the reaction mixture by 62, 67, and 36 %, respectively, however, 6-phosphogluconate dehydrogenase was insensitive to Cd²⁺. This revised version was published online in June 2006 with corrections to the Cover Date.     

Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

This study set out to identify novel gene variants that may contribute to human obesity, by combining human exosome sequencing analyses with systematic functional screening in Drosophila. This identifies a number of novel obesity-associated genes which control adiposity in flies, and uncovers a potential role for the Hippo signaling pathway in obesity.

Obesity is a major risk factor for type 2 diabetes, cardiovascular disease, cancers, and, most recently, COVID-19 [1]. Despite the obvious environmental drivers to weight gain, multiple genetic studies have demonstrated that 40% to 70% of the variation in body weight is attributable to genetic variation [2]. The discovery of genes that contribute to the regulation of human body weight can provide insights into the mechanisms involved in energy homeostasis and identify potential targets for weight loss therapy. Moreover, drug targets supported by human genetic evidence are more likely to transit successfully through the drug discovery pipeline [3].A classical approach to the discovery of pathogenic variants is to investigate consanguineous populations with high degrees of parental relatedness (parents who are first or second cousins) where large portions of the genome are identical by descent as a result of family structure in preceding generations (long regions of homozygosity). Indeed, studies in consanguineous families led to the discovery of the first homozygous loss-of-function mutations in the genes encoding leptin (LEP; [4]) and the leptin receptor (LEPR; [5]) associated with severe obesity. However, at the time, the function of leptin and its receptor had already been established in ob/ob and db/db mice, respectively [6], so the pathogenicity of homozygous mutations that resulted in loss of function in cells was readily established.The situation is more complex when studying homozygous mutations in new candidate genes. Some of these genes may play a direct causal role in the development of obesity, others may increase susceptibility to obesity only in certain contexts, and some genes will play no role at all. Recent large-scale studies in healthy people in outbred populations have revealed that a significant proportion of rare homozygous variants that are predicted to cause a loss of function do not result in a clinically discernible phenotype [7,8]. As such, identifying the subset of genes that may be involved in the regulation of adiposity in large human genetic studies presents a major hurdle.For some diseases, functional screens in cultured cells permit rapid testing of candidate genes, as exemplified by studies of insulin secretion in islet cells for genes associated with type 2 diabetes [9]. However, obesity is a systems-level disorder that cannot be replicated in cells. As such, a functional screen in vivo is needed. Here, we use Drosophila to screen the functional consequences of knocking down expression of candidate human obesity genes and to explore the complex interactions between multiple organ systems that are regulated by environmental and genetic factors.Drosophila has been a useful tool in the functional characterisation of human disease-associated genes [10–12]. Many organ systems and metabolic enzymes are highly conserved in Drosophila, as are the major regulatory mechanisms involved in metabolic homeostasis [13,14]. As in humans, Drosophila accumulate lipids and become obese when raised on a high-fat or high-sugar diet, developing cardiomyopathy and diabetic phenotypes [15,16]. Furthermore, more than 60% of the genes identified in an unbiased genome-wide RNAi screen for increased fat levels in Drosophila have human orthologues [17]. Most studies in Drosophila have performed forward genetic screens resulting in obesity [18] before assessing whether misregulation of the corresponding mammalian orthologue affects adiposity [17]. Another report knocked down Drosophila orthologs of human genes near body mass index (BMI) loci from GWAS studies to identify genes regulating adiposity [19].Here, instead, we chose to take advantage of new data from a cohort of patients carrying rare genetic variants that might cause severe early-onset obesity. We set out to identify, in Drosophila, whether any of these genes are likely to be responsible for the obese phenotype. An additional advantage of working with Drosophila is the potential to identify interacting genes and signalling pathways. We proposed that it would then be possible to search for variants in human orthologues of these genes in larger cohorts of patients, to discover further as yet unidentified genes regulating human obesity.To increase our chances of finding pathogenic variants, we focused on rare homozygous variants identified in probands with severe obesity, many from consanguineous families. After knocking down expression of Drosophila orthologues of candidate human obesity genes, we discovered 4 genes that significantly increased triacylglyceride (TAG) levels. Importantly, none of these genes had been associated previously with human obesity, but the pathways in which they act are known and could be further analysed in Drosophila. Knockdown of further members of one of these signalling pathways, the Hippo pathway, also gave an obesity phenotype, highlighting the success of our approach. We then searched for variants in the novel obesity genes we identified in Drosophila, and their associated signalling pathways, in larger cohorts of unrelated obese people and healthy controls. This uncovered yet another gene, which, when knocked down in Drosophila, increased adiposity. We demonstrate that the cross-fertilisation of human and Drosophila genetics is a powerful system to provide novel insights into the genetic and cellular processes regulating adiposity and may ultimately contribute to strategies for the prevention and treatment of obesity.     

Deciphering the mechanism behind the varied binding activities of COXIBs through Molecular Dynamic Simulations,MM-PBSA binding energy calculations and per-residue energy decomposition studies

COX-2 is a well-known drug target in inflammatory disorders. COX-1/COX-2 selectivity of NSAIDs is crucial in assessing the gastrointestinal side effects associated with COX-1 inhibition. Celecoxib, rofecoxib, and valdecoxib are well-known specific COX-2 inhibiting drugs. Recently, polmacoxib, a COX-2/CA-II dual inhibitor has been approved by the Korean FDA. These COXIBs have similar structure with diverse activity range. Present study focuses on unraveling the mechanism behind the 10-fold difference in the activities of these sulfonamide-containing COXIBs. In order to obtain insights into their binding with COX-2 at molecular level, molecular dynamics simulations studies, and MM-PBSA approaches were employed. Further, per-residue decomposition of these energies led to the identification of crucial amino acids and interactions contributing to the differential binding of COXIBs. The results clearly indicated that Leu338, Ser339, Arg499, Ile503, Phe504, Val509, and Ser516 (Leu352, Ser353, Arg513, Ile517, Phe518, Val523, and Ser530 in PGHS-1 numbering) were imperative in determining the activity of these COXIBs. The binding energies and energy contribution of various residues were similar in all the three simulations. The results suggest that hydrogen bond interaction between the hydroxyl group of Ser516 and five-membered ring of diarylheterocycles augments the affinity in COXIBs. The SAR of the inhibitors studied and the per-residue energy decomposition values suggested the importance of Ser516. Additionally, the positive binding energy obtained with Arg106 explains the binding of COXIBs in hydrophobic channel deep in the COX-2 active site. The findings of the present work would aid in the development of potent COX-2 inhibitors.     

Structural and biophysical properties of h-FANCI ARM repeat protein

Fanconi anemia complementation groups – I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein–protein and protein–DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.     

Bacterial Virulence Factors: Secreted for Survival

Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. These factors are either secretory, membrane associated or cytosolic in nature. The cytosolic factors facilitate the bacterium to undergo quick adaptive—metabolic, physiological and morphological shifts. The membrane associated virulence factors aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors act synergistically to kill the host cells. In this review, we revisit the role of some of the secreted virulence factors of two human pathogens: Mycobacterium tuberculosis—an intracellular pathogen and Bacillus anthracis—an extracellular pathogen. The advances in research on the role of secretory factors of these pathogens during infection are discussed.     

Role of Se(VI) in counteracting oxidative damage in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. under Cr(VI) stress

Chromium (Cr) is considered to be one of the major environmental hazards and poses a threat to both plant and animal health. Selenium (Se), however, has been recognized as an essential micronutrient in plants. To understand the role of Se(VI) in oxidative stress management and regulation of antioxidative defence mechanism against heavy metal stress, the seedlings of Brassica juncea L. were raised in Petri plates containing nutrient media supplemented with only with Se(VI) and Cr(VI), or their combination. It was observed that of Cr(VI) causes an increase in reactive oxygen species (ROS) in the seedlings leading to oxidative stress. Histological studies using confocal and visible microscopy confirmed the biochemical results. Supplementation of up to 4 µM of Se(VI) to media containing 300 µM of Cr(VI) reduced the contents of ROS and increased enzymatic and non-enzymatic antioxidants in the seedlings. At a concentration of 6 µM, however, Se(VI) was toxic. The results suggested that at appropriate concentrations, the exogenous application of Se(VI) enabled the B. juncea seedlings to counteract the effects of Cr(VI), thereby increasing the resistance of plants.     

Isolation of a Halophilic,Bacteriorhodopsin-producing Archaeon,Haloferax larsenii RG3D.1 from the Rocky Beach of Malvan,West Coast of India

Haloarchaea live in saline environments and require salt for their growth. The present study reports on the isolation of a haloarchaeal strain RG3D.1 identified as Haloferax larsenii using Sehgal and Gibbons medium with 3.42 M NaCl, from Rock Garden at Malvan in the West Coast of India. The rock type of Malvan Coast is correlated based on geological setting with the basal member of the Bababudan group placed at 2728 ± 102 million years ago (Ma). The haloarchaeal strain produced bacteriorhodopsin (BR) (0.137 g l^?1) and generated electric potential of 49.2 mV when exposed to sunlight. Thus, this is the first report on the isolation of a haloarchaeon, Haloferax larsenii, from red rocky substrate and production of BR.