Localisation and function of key axonemal microtubule inner proteins and dynein docking complex members reveal extensive diversity among vertebrate motile cilia.

Vertebrate motile cilia are classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition. The extent to which these features are conserved in (9+0) cilia remains unclear. CFAP53, a key axonemal filamentous microtubule inner protein (fMIP) and a centriolar satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, CFAP53 functions redundantly with a paralogous fMIP, MNS1. MNS1 localises to ciliary axonemes, and combined loss of both proteins in zebrafish and mice caused severe outer dynein arm loss from (9+2) cilia, significantly affecting their motility. Using immunoprecipitation, we demonstrate that, whereas MNS1 can associate with itself and CFAP53, CFAP53 is unable to self-associate. We also show that additional axonemal dynein-interacting proteins, two outer dynein arm docking (ODAD) complex members, show differential localisation between types of motile cilia. Together, our findings clarify how paralogous fMIPs, CFAP53 and MNS1, function in regulating (9+2) versus (9+0) cilia motility, and further emphasise extensive structural diversity among these organelles.

The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

MITOL-mediated DRP1 ubiquitylation and degradation promotes mitochondrial hyperfusion in a CMT2A-linked MFN2 mutant.

Mutations in mitofusin 2 (MFN2) that are associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. One such abundant MFN2 mutation, R364W, results in the generation of elongated, interconnected mitochondria. However, the mechanism leading to this mitochondrial aberration remains poorly understood. Here, we show that mitochondrial hyperfusion in the presence of R364W-MFN2 is due to increased degradation of DRP1 (also known as DNM1L). The E3 ubiquitin ligase MITOL (also known as MARCHF5) is known to ubiquitylate both MFN2 and DRP1. Interaction with and subsequent ubiquitylation by MITOL is stronger in the presence of wild-type MFN2 than with R364W-MFN2. This differential interaction of MITOL with MFN2 in the presence of R364W-MFN2 renders the ligase more available for DRP1 ubiquitylation. Multi-monoubiquitylation and proteasomal degradation of DRP1 in R364W-MFN2 cells in the presence of MITOL eventually leads to mitochondrial hyperfusion. Here, we provide a mechanistic insight into mitochondrial hyperfusion, while also reporting that MFN2 can indirectly modulate DRP1 - an effect not shown previously. This article has an associated First Person interview with the first author of the paper.

Oncogene-mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

Variability in the Responses of Hepatitis B Virus D-Subgenotypes to Antiviral Therapy: Designing Pan-D-Subgenotypic Reverse Transcriptase Inhibitors.

Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

CMT2A-linked mitochondrial hyperfusion-driving mutant MFN2 perturbs ER-mitochondrial associations and Ca2+ homeostasis.

BACKGROUND INFORMATION: Mitofusin2 (MFN2), an important molecular player that regulates mitochondrial fusion, also helps maintain the inter-organellar contact sites, referred as mitochondria associated membranes (MAMs) that exist between the ER and mitochondria. The study deals with a mutant of MFN2, R364W-MFN2, linked with the neuropathy, Charcot Marie Tooth (CMT) disease. Previous studies show that this mutant promotes mitochondrial hyperfusion. Here, we try to decipher the role of R364W-MFN2 in affecting the ER mitochondrial associations at the MAM junctions and inter-organellar calcium signalling between the ER and the mitochondria. RESULTS: Our results show that R364W-MFN2 altered ER-mitochondria association at the MAM junctions, predisposed mitochondria towards cellular stress with the mitochondria undergoing rapid fission upon induction of mild stress and perturbs inter-organellar calcium homeostasis. CONCLUSION: The results indicate that R364W-MFN2 not only affects mitochondrial morphology and dynamics but also modulate its interaction with the ER and Ca2+ signalling between the two organelles. SIGNIFICANCE: This study provides significant insight that presence of the R364W-MFN2 mutation makes cells susceptible towards stress, thus negatively affecting cellular health which altogether might culminate in the form of the CMT neuropathy.

Cytosolic aggregates in presence of non-translocated proteins perturb endoplasmic reticulum structure and dynamics.

Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non-translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non-translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three-way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins-reticulons, and altered distribution of those associated with the three-way ER junctions-Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non-translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven-pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non-translocated precursor proteins-their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.

A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate.

The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.

Whole-exome analyses of congenital muscular dystrophy and congenital myopathy patients from India reveal a wide spectrum of known and novel mutations.

BACKGROUND AND PURPOSE: Congenital muscular dystrophies (CMDs) and congenital myopathies (CMs) are a group of genetically and clinically heterogeneous degenerative primary muscle disorders with onset at birth or during infancy. Due to vast heterogeneity, clinical examination and protein-based analyses often fail to identify the genetic causes of these diseases. The aim of this study was to genetically diagnose a cohort of 36 difficult-to-diagnose CMD and CM cases of Indian origin using next-generation sequencing methods. METHODS: Whole-exome sequencing (WES) was performed to identify pathogenic mutations in previously reported CMD and CM-related genes using variant calling and stringent variant filtration process. Subsequently, in silico homology modelling and molecular dynamics simulations (MDS) studies were undertaken for a number of novel and missense variants. RESULTS: A total of 33 and 21 rare and deleterious mutations were identified in 28 genes previously reported in CMD and CM based on OMIM, ClinVar and Orphanet, respectively. We could accurately diagnose 54% patients (n = 12/22) in the CMD group and 35% patients (n = 5/14) in the CM group. Furthermore, MDS studies for mutations located in LMNA, LAMA2 and RYR1 suggest that the wild-type proteins are more stable than their mutant counterparts, implying a potential mechanism of pathogenesis. CONCLUSION: The WES findings led us to identify reported as well as novel variants for the first time in Indian patients with CMD and CM. This allowed us to achieve an accurate genetic diagnosis, which was difficult using conventional diagnostic tools. Transferring these WES findings to clinical practice will help guide clinical care of the affected patients and inform genetic counselling.

Supercritical carbon dioxide extracts of small cardamom and yellow mustard seeds have fasting hypoglycaemic effects: diabetic rat, predictive iHOMA2 models and molecular docking study.

In the present investigation, the supercritical carbon dioxide (SC-CO2) extracts of small cardamom (SC) and yellow mustard (YM) seeds have been investigated for their efficacies in combating type 2 diabetes in streptozotocin-induced Wistar albino rats. Fasting blood glucose (FBG) levels in the rats were monitored on days 8, 15 and 21. On day 15, FBG level reduced appreciably by 31·49 % in rats treated with SC seed extract and by 32·28 % in rats treated with YM seed extract, comparable to metformin (30·70 %) and BGR-34 (a commercial polyherbal drug) (31·81 %) administered rats. Either extract exhibited desirable effects on hepatic glucose-6-phosphatase, glucose-6-phosphate dehydrogenase (G6PD) and catalase activities in controlling diabetes. A molecular docking exercise was conducted to identify specific compounds in the extracts which possessed augmenting effect on G6PD. The results revealed that all the bioactive compounds in the extracts have binding affinities with the enzyme and contributed to the antidiabetic efficacies of the extracts as G6PD augmenters. The effects of the extracts on insulin sensitivity and glucose uptake were investigated using non-invasive modelling by iHOMA2 software. This in vitro approach indicated that extract administration resulted in increased both insulin sensitivity of the liver and glucose uptake in the gut. The findings of the present study attest these SC-CO2 extracts of the spices as safe alternatives of metformin and BGR-34 in combating type 2 diabetes and could be safely subjected to clinical studies. These extracts could also be employed in designing proactive food supplements in mitigating the metabolic disorder.

PresRAT: a server for identification of bacterial small-RNA sequences and their targets with probable binding region.

Bacterial small-RNA (sRNA) sequences are functional RNAs, which play an important role in regulating the expression of a diverse class of genes. It is thus critical to identify such sRNA sequences and their probable mRNA targets. Here, we discuss new procedures to identify and characterize sRNA and their targets via the introduction of an integrated online platform 'PresRAT'. PresRAT uses the primary and secondary structural attributes of sRNA sequences to predict sRNA from a given sequence or bacterial genome. PresRAT also finds probable target mRNAs of sRNA sequences from a given bacterial chromosome and further concentrates on the identification of the probable sRNA-mRNA binding regions. Using PresRAT, we have identified a total of 66,209 potential sRNA sequences from 292 bacterial genomes and 2247 potential targets from 13 bacterial genomes. We have also implemented a protocol to build and refine 3D models of sRNA and sRNA-mRNA duplex regions and generated 3D models of 50 known sRNAs and 81 sRNA-mRNA duplexes using this platform. Along with the server part, PresRAT also contains a database section, which enlists the predicted sRNA sequences, sRNA targets, and their corresponding 3D models with structural dynamics information.

Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

Connecting signaling and metabolic pathways in EGF receptor-mediated oncogenesis of glioblastoma.

As malignant transformation requires synchronization of growth-driving signaling (S) and metabolic (M) pathways, defining cancer-specific S-M interconnected networks (SMINs) could lead to better understanding of oncogenic processes. In a systems-biology approach, we developed a mathematical model for SMINs in mutated EGF receptor (EGFRvIII) compared to wild-type EGF receptor (EGFRwt) expressing glioblastoma multiforme (GBM). Starting with experimentally validated human protein-protein interactome data for S-M pathways, and incorporating proteomic data for EGFRvIII and EGFRwt GBM cells and patient transcriptomic data, we designed a dynamic model for EGFR-driven GBM-specific information flow. Key nodes and paths identified by in silico perturbation were validated experimentally when inhibition of signaling pathway proteins altered expression of metabolic proteins as predicted by the model. This demonstrated capacity of the model to identify unknown connections between signaling and metabolic pathways, explain the robustness of oncogenic SMINs, predict drug escape, and assist identification of drug targets and the development of combination therapies.

Calcium dependent regulation of protein ubiquitination - Interplay between E3 ligases and calcium binding proteins.

The ubiquitination status of proteins and intracellular calcium levels are two factors which keep changing inside any living cell. These two events appear to be independent of each other but recent experimental evidences show that ubiquitination of cellular proteins are influenced by calcium, Calmodulin, Calmodulin-dependent kinase II and other proteins of calcium dependent pathways. E3 ligases like Nedd4, SCF complex, APC, GP78 and ITCH are important regulators of calcium mediated processes. A bioinformatics analysis to inspect sequences and interacting partners of 242 candidate E3 ligases show the presence of calcium and/or Calmodulin binding motifs/domains within their sequences. Building a protein-protein interaction (PPI) network of human E3 ligase proteins identifies Ca2+ related proteins as direct interacting partners of E3 ligases. Review of literature, analysis of E3 ligase sequences and their interactome suggests an interconnectivity between calcium signaling and the overall UPS system, especially emphasizing that a subset of E3 ligases have importance in physiological pathways modulated by calcium.

Hedgehog Antagonist Pyrimidine-Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation.

One of the crucial regulators of embryonic patterning and tissue development is the Hedgehog-glioma (Hh-Gli) signalling pathway; its uncontrolled activation has been implicated in different types of cancer in adult tissues. Primary cilium is one of the important factors required for the activation of Hh signalling, as it brings the critical components together for key protein-protein interactions required for Hh pathway regulation. Most of the synthetic and natural small molecule modulators of the pathway primarily antagonise Smoothened (Smo) or other effectors like Hh ligand or Gli. Here, we report a previously described Hh antagonist, with a pyrimidine-indole hybrid (PIH) core structure, as an inhibitor of ciliogenesis. The compound is unique in its mode of action, as it shows perturbation of microtubule dynamics in both cell-based assays and in vivo systems (zebrafish embryos). Further studies revealed that the probable targets are α-tubulin and its acetylated form, found in the cytoplasm and primary cilia. PIH also showed axonal defasiculation in developing zebrafish embryos. We thus propose that PIH antagonises Hh signalling by repressing cilia biogenesis and disassembling α-tubulin from its stabilised form.

Quinoline-Glycomimetic Conjugates Reducing Lipogenesis and Lipid Accumulation in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by excess accumulation of triglyceride in hepatocytes, is the major cause of chronic liver disease worldwide and no approved drug is available. The mechanistic target of rapamycin (mTOR) complexes has been implicated in promoting lipogenesis and fat accumulation in the liver, and thus, serve as attractive drug targets. The generation of non- or low cytotoxic mTOR inhibitors is required because existing cytotoxic mTOR inhibitors are not useful for NAFLD therapy. New compounds based on the privileged adenosine triphosphate (ATP) site binder quinoline scaffold conjugated to glucose and galactosamine derivatives, which have significantly low cytotoxicity, but strong mTORC1 inhibitory activity at low micromolar concentrations, have been synthesized. These compounds also effectively inhibit the rate of lipogenesis and lipid accumulation in cultured hepatocytes. This is the first report of glycomimetic-quinoline derivatives that reduce lipid load in hepatocytes.

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas.

Initiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the Escherichia coli model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.

Enhanced basepair dynamics pre-disposes protein-assisted flips of key bases in DNA strand separation during transcription initiation.

Localized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes, including transcription and replication. Little is known about how these strand separations occur. The strand-separated E.coli RNA polymerase-promoter open-complex structure showed four bases of the non-template strand, the master base -11A, -7, -6 and +2, in a flipped state and inserted into protein pockets. To explore whether any property of these bases in the duplex state pre-disposes them to flipping, NMR studies were performed on a wild-type promoter in the duplex state. Measurement of relaxation times indicates that a limited number of base pairs, including the flipped ones, have faster opening rates than the rest. Molecular dynamics studies also show an inherently high dynamic character of the -11A:T base pair in the wild-type strand-paired state. In order to explore the role of the RNA polymerase in the flipping process, we have used 2-aminopurine as a fluorescent probe. Slower kinetics of the increase of 2-aminopurine fluorescence was observed with RNA polymerases containing several mutant σ70s. This may be interpreted as the protein playing an important role in enhancing the flipping rate. These results suggest that flipping of -11A, and perhaps other flipped bases observed in the open-complex, is facilitated by its inherent proclivity to open-up with further assistance from the protein, thus leading to a strand-open state. Other DNA-based processes that require strand-separation may use similar pathways for strand separation. We conclude that not only basepair stability, but also dynamics may play an important role in the strand-separation.

A survey on prediction of specificity-determining sites in proteins.

Specificity-determining sites (SDS) are the key positions of a protein family that show a specific conservation of amino acids, related to the subfamily members of that family. SDS play crucial role in developing functional variation within the protein family during the course of evolution. Thus, it is important to identify SDS to understand the evolutionary process of diversification of biological functions within a protein family. A wide range of computational tools have been designed to detect such SDS. In this review, we intend to examine the concept of SDS in more details along with the advancements and drawbacks of different computational approaches designed towards successful prediction of SDS. Further, we discussed the algorithms behind the computational approaches developed till date and provide an exhaustive comparison of performance of each method. We also introduce a new ensemble approach, SubSite as another tool to predict SDS through a user-friendly webserver available at www.hpppi.iicb.res.in/subsite.