Rajan Vyas

Abstract: Mutant Huntingtin (mtHtt) causes neurodegeneration in Huntington's disease (HD) by evoking defects in the mitochondria, but the underlying mechanisms remains elusive. Our proteomic analysis identifies valosin-containing protein (VCP) as an mtHtt-binding protein on the mitochondria. Here we show that VCP is selectively translocated to the mitochondria, where it is bound to mtHtt in various HD models. Mitochondria-accumulated VCP elicits excessive mitophagy, causing neuronal cell death. Blocking mtHtt/VCP mitochondrial interaction with a peptide, HV-3, abolishes VCP translocation to the mitochondria, corrects excessive mitophagy and reduces cell death in HD mouse- and patient-derived cells and HD transgenic mouse brains. Treatment with HV-3 reduces behavioural and neuropathological phenotypes of HD in both fragment- and full-length mtHtt transgenic mice. Our findings demonstrate a causal role of mtHtt-induced VCP mitochondrial accumulation in HD pathogenesis and suggest that the peptide HV-3 might be a useful tool for developing new therapeutics to treat HD.

Abstract: The range of antibiotic therapy for the control of bacterial infections is becoming increasingly limited because of the rapid rise in multidrug resistance in clinical bacterial isolates. A few diseases, such as tuberculosis, which were once thought to be under control, have re-emerged as serious health threats. These problems have resulted in intensified research to look for new inhibitors for bacterial pathogens. Of late, the peptidoglycan (PG) layer, the most important component of the bacterial cell wall has been the subject of drug targeting because, first, it is essential for the survivability of eubacteria and secondly, it is absent in humans. The last decade has seen tremendous inputs in deciphering the 3-D structures of the PG biosynthetic enzymes. Many inhibitors against these enzymes have been developed using virtual and high throughput screening techniques. This review discusses the mechanistic and structural properties of the PG biosynthetic enzymes and inhibitors developed in the last decade.

Abstract: One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson-Crick base pair with correct deoxycytidine 5'-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5'-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson-Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg(2+) appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3'-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.

Abstract: Imidazoleglycerol-phosphate dehydratase (IGPD; HisB), which catalyses the conversion of imidazoleglycerol-phosphate (IGP) to imidazoleacetol-phosphate in the histidine biosynthesis pathway, is absent in mammals. This feature makes it an attractive target for herbicide discovery. Here, the crystal structure of Mycobacterium tuberculosis (Mtb) IGPD is reported together with the first crystal structures of substrate-bound and inhibited (by 3-amino-1,2,4-triazole; ATZ) forms of IGPD from any organism. The overall tertiary structure of Mtb IGPD, a four-helix-bundle sandwiched between two four-stranded mixed β-sheets, resembles the three-dimensional structures of IPGD from other organisms; however, Mtb IGPD possesses a unique structural feature: the insertion of a one-turn 310-helix followed by a loop ten residues in length. The functional form of IGPD is 24-meric, exhibiting 432 point-group symmetry. The structure of the IGPD–IGP complex revealed that the imidazole ring of the IGP is firmly anchored between the two Mn atoms, that the rest of the substrate interacts through hydrogen bonds mainly with residues Glu21, Arg99, Glu180, Arg121 and Lys184 which protrude from three separate protomers and that the 24-mer assembly contains 24 catalytic centres. Both the structural and the kinetic data demonstrate that the inhibitor 3-amino-1,2,4-triazole inhibits IGPD competitively.

Abstract: Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase. Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

Abstract: The innate immune system constitutes the first line of defence against invading microbes. The basis of this defence resides in the recognition of defined structural motifs of the microbes called "Microbial associated molecular patterns" that are absent in the host. Cell wall, the outer layer of both bacterial and fungal cells, a unique structure that is absent in the host and is recognized by the germ line encoded host receptors. Nucleotide oligomerization domain proteins, peptidoglycan recognition proteins and C-type lectins are host receptors that are involved in the recognition of bacterial cell wall (usually called peptidoglycan), whereas fungal cell wall components (N- and O-linked mannans, β-glucans etc.) are recognized by host receptors like C-type lectins (Dectin-1, Dectin-2, mannose receptor, DC-SIGN), Toll like receptors-2 and -4 (TLR-2 and TLR-4). These recognitions lead to activation of a variety of host signaling cascades and ultimate production of anti-microbial compounds including phospholipase A2, antimicrobial peptides, lysozyme, reactive oxygen and nitrogen species. These molecules act in cohort against the invading microbes to eradicate infections. Additionally pathogen recognition leads to the production of cytokines, which further activate the adaptive immune system. Both pathogenic and commensal bacteria and fungus use numerous strategies to subvert the host defence. These strategies include bacterial peptidoglycan glycan backbone modifications by O-acetylation, N-deacetylation, N-glycolylation and stem peptide modifications by amidation of meso-Diaminopimelic acid; fungal cell wall modifications by shielding the β-glucan layer with mannoproteins and α-1,3 glucan. This review focuses on the recent advances in understanding the role of bacterial and fungal cell wall in their innate immune recognition and evasion strategies.

Abstract: It is generally assumed that an enzyme-substrate (ES) complex contains all components necessary for catalysis and that conversion to products occurs by rearrangement of atoms, protons, and electrons. However, we find that DNA synthesis does not occur in a fully assembled DNA polymerase–DNA–deoxynucleoside triphosphate complex with two canonical metal ions bound. Using time-resolved x-ray crystallography, we show that the phosphoryltransfer reaction takes place only after the ES complex captures a third divalent cation that is not coordinated by the enzyme. Binding of the third cation is incompatible with the basal ES complex and requires thermal activation of the ES for entry. It is likely that the third cation provides the ultimate boost over the energy barrier to catalysis of DNA synthesis.

Abstract: In the last few years, increased emphasis has been devoted to understanding the contribution of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) to human pathology in general, and neurodegenerative diseases in particular. A major reason for this is the central role that this subdomain of the ER plays in metabolic regulation and in mitochondrial biology. As such, aberrant MAM function may help explain the seemingly unrelated metabolic abnormalities often seen in neurodegeneration. In the specific case of Alzheimer disease (AD), besides perturbations in calcium and lipid homeostasis, there are numerous documented alterations in mitochondrial behavior and function, including reduced respiratory chain activity and oxidative phosphorylation, increased free radical production, and altered organellar morphology, dynamics, and positioning (especially perinuclear mitochondria). However, whether these alterations are primary events causative of the disease, or are secondary downstream events that are the result of some other, more fundamental problem, is still unclear. In support of the former possibility, we recently reported that C99, the C-terminal processing product of the amyloid precursor protein (APP) derived from its cleavage by β-secretase, is present in MAM, that its level is increased in AD, and that this increase reduces mitochondrial respiration, likely via a C99-induced alteration in cellular sphingolipid homeostasis. Thus, the metabolic disturbances seen in AD likely arise from increased ER-mitochondrial communication that is driven by an increase in the levels of C99 at the MAM.

Abstract: In recent years, several important advances have been made in our understanding of the pathways that lead to cell dysfunction and death in Parkinson's disease (PD) and Huntington's disease (HD). Despite distinct clinical and pathological features, these two neurodegenerative diseases share critical processes, such as the presence of misfolded and/or aggregated proteins, oxidative stress, and mitochondrial anomalies. Even though the mitochondria are commonly regarded as the "powerhouses" of the cell, they are involved in a multitude of cellular events such as heme metabolism, calcium homeostasis, and apoptosis. Disruption of mitochondrial homeostasis and subsequent mitochondrial dysfunction play a key role in the pathophysiology of neurodegenerative diseases, further highlighting the importance of these organelles, especially in neurons. The maintenance of mitochondrial integrity through different surveillance mechanisms is thus critical for neuron survival. Mitochondria display a wide range of quality control mechanisms, from the molecular to the organellar level. Interestingly, many of these lines of defense have been found to be altered in neurodegenerative diseases such as PD and HD. Current knowledge and further elucidation of the novel pathways that protect the cell through mitochondrial quality control may offer unique opportunities for disease therapy in situations where ongoing mitochondrial damage occurs. In this review, we discuss the involvement of mitochondrial dysfunction in neurodegeneration with a special focus on the recent findings regarding mitochondrial quality control pathways, beyond the classical effects of increased production of reactive oxygen species (ROS) and bioenergetic alterations. We also discuss how disturbances in these processes underlie the pathophysiology of neurodegenerative disorders such as PD and HD.