Characterization of Kiss/Kissr system and expression profiling through developmental stages indicate kiss1 to be the active isotype in Clarias magur.

Kisspeptin (Kiss) and kisspeptin receptor (Kissr) system is a key regulator of GnRH expression in several vertebrates. The Indian catfish, Clarias magur, is popular in the Indian sub-continent, and a neo-type of the Asian catfish, C. batrachus. Catfish breeding is constrained as males do not release milt captivity with/without stimulation. Magur Kiss/Kissr system comprising of kiss1, kiss2, kissr1, and kissr2 genes was characterized for the first time. Full-length mRNA was sequenced using RACE PCR. Neighbor-joining tree of predicted proteins shows one clade of teleost orthologs. Magur whole genome (NCBI GenBank) has single copies of each gene, though yet unannotated/misannotated. Anomalies in the nomenclature of earlier sequences in GenBank were noted. Relative gene expression was profiled during various ontogenic stages, in six tissues including brain and gonads at maturity, and also in brains and gonads of premature and spent fish. Expression of gnrh1, gnrhr1, and gnrhr2 was estimated concomitantly. The kiss1 was the first to be twofold upregulated (P < 0.05) at 12 h post fertilization. Kiss/Kissr genes expressed primarily in the brain, ovary, and testis. Though kiss2 was 10 times higher than kiss1, only kiss1 showed significant modulation across stages and appears to be the active isotype that regulates GnRH in magur.

Structure-function analyses reveal Arabidopsis thaliana HDA7 to be an inactive histone deacetylase.

Histone deacetylases (HDACs), responsible for the removal of acetyl groups from histone tails, are important epigenetic factors. They play a critical role in the regulation of gene expression and are significant in the context of plant growth and development. The Rpd3/Hda1 family of HDACs is reported to regulate key biological processes in plants, such as stress response, seed, embryonic, and floral development. Here, we characterized Arabidopsis thaliana HDA7, a Class I, Rpd3/Hda1 family HDAC. SAXS and AUC results show that the recombinantly expressed and purified histone deacetylase domain of AtHDA7 exists as a monomer in solution. Further, the crystal structure showed AtHDA7 to fold into the typical α/ß arginase fold, characteristic of Rpd3/Hda1 family HDACs. Sequence analysis revealed that the Asp and His residues of the catalytic 'XDXH' motif present in functional Rpd3/Hda1 family HDACs are mutated to Gly and Pro, respectively, in AtHDA7, suggesting that it might be catalytically inactive. The Asp and His residues are important for Zn2+-binding. Not surprisingly, the crystal structure did not have Zn2+ bound in the catalytic pocket, which is essential for the HDAC activity. Further, our in vitro activity assay revealed AtHDA7 to be inactive as an HDAC. A search in the sequence databases suggested that homologs of AtHDA7 are found exclusively in the Brassicaceae family to which Arabidopsis belongs. It is possible that HDA7 descended from HDA6 through whole genome duplication and triplication events during evolution, as suggested in a previous phylogenetic study.

Crystal structure of the N-terminal domain of MtClpC1 in complex with the anti-mycobacterial natural peptide Lassomycin.

Antibiotics form our frontline therapy against disease-causing bacteria. Unfortunately, antibiotic resistance is becoming more common, threatening a future where these medications can no longer cure infections. Furthermore, the emergence of multidrug-resistant (MDR), totally drug-resistant (TDR), and extensively drug-resistant (XDR) tuberculosis has increased the urgency of discovering new therapeutic leads with unique modes of action. Some natural peptides derived from actinomycetes, such as Cyclomarin A, Lassomycin, Rufomycin I, and Ecumicin, have potent and specific bactericidal activity against Mycobacterium tuberculosis, with the specificity owing to the fact that these peptides target the ClpC1 ATPase, an essential enzyme in mycobacteria, and inhibit/activate the proteolytic activity of the ClpC1/P1/P2 complex that participates in protein homeostasis. Here, we report the high-resolution crystal structure of the N-terminal domain of ClpC1 (ClpC1 NTD) in complex with Lassomycin, showing the specific binding mode of Lassomycin. In addition, the work also compares the Lassomycin complex structure with the previously known structures of ClpC1 NTD in complex with other natural peptides such as Cyclomarin A, Rufomycin I, and Ecumicin.

The plant nucleoplasmin AtFKBP43 needs its extended arms for histone interaction.

The nucleoplasmin family of histone chaperones is a key player in governing the dynamic architecture of chromatin, thereby regulating various DNA-templated processes. The crystal structure of the N-terminal domain of Arabidopsis thaliana FKBP43 (AtFKBP43), an FK506-binding immunophilin protein, revealed a characteristic nucleoplasmin fold, thus confirming it to be a member of the FKBP nucleoplasmin class. Small-Angle X-ray Scattering (SAXS) analyses confirmed its pentameric nature in solution, and additional studies confirmed the nucleoplasmin fold to be highly stable. Unlike its homolog AtFKBP53, the AtFKBP43 nucleoplasmin core domain could not interact with histones and required the acidic arms, C-terminal to the core, for histone association. However, SAXS generated low-resolution envelope structure, ITC, and AUC results revealed that an AtFKBP43 pentamer with C-terminal extensions interacts with H2A/H2B dimer and H3/H4 tetramer in an equimolar ratio, like AtFKBP53. Put together, AtFKBP43 belongs to a hitherto unreported subclass of FKBP nucleoplasmins that requires the C-terminal acidic stretches emanating from the core domain for histone interaction.

Plant-specific HDT family histone deacetylases are nucleoplasmins.

Histone acetyltransferase (HAT)- and histone deacetylase (HDAC)-mediated histone acetylation and deacetylation regulate nucleosome dynamics and gene expression. HDACs are classified into different families, with HD-tuins or HDTs being specific to plants. HDTs show some sequence similarity to nucleoplasmins, the histone chaperones that aid in binding, storing, and loading H2A/H2B dimers to assemble nucleosomes. Here, we solved the crystal structure of the N-terminal domain (NTD) of all four HDTs (HDT1, HDT2, HDT3, and HDT4) from Arabidopsis (Arabidopsis thaliana). The NTDs form a nucleoplasmin fold, exist as pentamers in solution, and are resistant to protease treatment, high temperature, salt, and urea conditions. Structurally, HDTs do not form a decamer, unlike certain classical nucleoplasmins. The HDT-NTD requires an additional A2 acidic tract C-terminal to the nucleoplasmin domain for interaction with histone H3/H4 and H2A/H2B oligomers. We also report the in-solution structures of HDT2 pentamers in complex with histone oligomers. Our study provides a detailed structural and in vitro functional characterization of HDTs, revealing them to be nucleoplasmin family histone chaperones. The experimental confirmation that HDTs are nucleoplasmins may spark new interest in this enigmatic family of proteins.

MBZM-N-IBT, a Novel Small Molecule, Restricts Chikungunya Virus Infection by Targeting nsP2 Protease Activity In Vitro, In Vivo, and Ex Vivo.

The increase in disease incidences and persistent Chikungunya virus (CHIKV)-induced arthritis have been a huge burden on public health globally. In the absence of specific antivirals or vaccines, it is essential to continue efforts to develop effective anti-CHIKV strategies. Our previous study showing the in vitro anti-CHIKV potential of a novel molecule 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) encouraged us to further validate its efficacy. Here, the effect of MBZM-N-IBT was evaluated in vitro in RAW 264.7 cells, in vivo in C57BL/6 mice, and ex vivo in human peripheral blood mononuclear cells (hPBMCs). The study demonstrated that CHIKV infection was efficiently abrogated in RAW 264.7 cells (IC50 = 22.34 µM) with significant inhibition in viral proteins. The inhibition was effective in the postentry step, and MBZM-N-IBT predominately interfered in the early stages of CHIKV life cycle. It was further supported when the protease activity of CHIKV-nsP2 was hindered by the compound. Moreover, it diminished the CHIKV-induced inflammatory responses in vitro through significant downregulation of all the major mitogen-activated protein kinases (MAPKs), NF-κB, cyclooxygenase (COX)-2, and cytokines. Furthermore, MBZM-N-IBT restricted CHIKV infection and inflammation in vivo, leading to reduced clinical scores and complete survival of C57BL/6 mice. Additionally, it has been noticed that the CHIKV infection was reduced remarkably in hPBMC-derived monocyte-macrophage populations ex vivo by the compound. In conclusion, it can be suggested that this novel compound MBZM-N-IBT has been demonstrated to be a potential anti-CHIKV molecule in vitro, in vivo, and ex vivo and fulfilled all the criteria to investigate further for successful treatment of CHIKV infection.

Crystal packing reveals rapamycin-mediated homodimerization of an FK506-binding domain.

Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes.

SSTP1, a Host Defense Peptide, Exploits the Immunomodulatory IL6 Pathway to Induce Apoptosis in Cancer Cells.

While the immunomodulatory pathways initiated in immune cells contribute to therapeutic response, their activation in cancer cells play a role in cancer progression. Also, many of the aberrantly expressed immunomodulators on cancer cells are considered as therapeutic targets. Here, we introduce host defense peptide (HDP), a known immuomodulator, as a therapeutic agent to target them. The cationic host defense peptides (HDPs), an integral part of the innate immune system, possess membranolytic activity, which imparts antimicrobial and antitumor efficacy to it. They act as immunomodulators by activating the immune cells. Though their antimicrobial function has been recently reassigned to immunoregulation, their antitumor activity is still attributed to its membranolytic activity. This membrane pore formation ability, which is proportional to the concentration of the peptide, also leads to side effects like hemolysis, limiting their therapeutic application. So, despite the identification of a variety of anticancer HDPs, their clinical utility is limited. Though HDPs are shown to exert the immunomodulatory activity through specific membrane targets on immune cells, their targets on cancer cells are unknown. We show that SSTP1, a novel HDP identified by shotgun cloning, binds to the active IL6/IL6Rα/gp130 complex on cancer cells, rearranging the active site residues. In contrast to the IL6 blockers inhibiting JAK/STAT activity, SSTP1 shifts the proliferative IL6/JAK/STAT signaling to the apoptotic IL6/JNK/AP1 pathway. In IL6Rα-overexpressing cancer cells, SSTP1 induces apoptosis at low concentration through JNK pathway, without causing significant membrane disruption. We highlight the importance of immunomodulatory pathways in cancer apoptosis, apart from its established role in immune cell regulation and cancer cell proliferation. Our study suggests that identification of the membrane targets for the promising anticancer HDPs might lead to the identification of new drugs for targeted therapy.

Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA.

Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure-function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility.

Structural analyses of PCNA from the fungal pathogen Candida albicans identify three regions with species-specific conformations.

An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. DATABASES: The PDB and SASBDB accession codes for CaPCNA are 7BUP and SASDHQ9, respectively.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters.

The peptidoglycan (PG) layer is an intricate and dynamic component of the bacterial cell wall, which requires a constant balance between its synthesis and hydrolysis. FtsEX complex present on the inner membrane is shown to transduce signals to induce PG hydrolysis. FtsE has sequence similarity with the nucleotide-binding domains (NBDs) of ABC transporters. The NBDs in most of the ABC transporters couple ATP hydrolysis to transport molecules inside or outside the cell. Also, this reaction cycle is driven by the dimerization of NBDs. Though extensive studies have been carried out on the Escherchia coli FtsEX complex, it remains elusive regarding how FtsEX complex helps in signal transduction or transportation of molecules. Also, very little is known about the biochemical properties and ATPase activities of FtsE. Because of its strong interaction with the membrane-bound protein FtsX, FtsE stays insoluble upon overexpression in E. coli, and thus, most studies on E. coli FtsE (FtsEEc) in the past have used refolded FtsE. Here in the present paper, for the first time, we report the soluble expression, purification, and biochemical characterization of FtsE from E. coli. The purified soluble FtsE exhibits high thermal stability, exhibits ATPase activity and has more than one ATP-binding site. We have also demonstrated a direct interaction between FtsE and the cytoplasmic loop of FtsX. Together, our findings suggest that during bacterial division, the ATPase cycle of FtsE and its interaction with the FtsX cytoplasmic loop may help to regulate the PG hydrolysis at the mid cell.

Structure analysis of thymidylate synthase from white spot syndrome virus reveals WSSV-specific structural elements.

White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures.

Recombinant expression, purification and SAXS analysis of Arabidopsis thaliana ClpC1.

Caseinolytic protease-associated chaperones (Clp chaperones) are HSP100 proteins belonging to the family of ATPases having diverse cellular functions, and they occur in various organisms ranging from bacteria to plants and mammals. Most Clp chaperones have a hexameric organization and associate with tetradecameric Clp proteases to recognize and unfold protein substrates that get degraded within the cellular milieu. Vascular plants have a diverse family of Clp chaperones compared to other organisms; wherein, the chloroplasts of Arabidopsis thaliana alone contain four distinct Clp chaperones, such as ClpC1, ClpC2, ClpD, and ClpB3. The paralogs AtClpC1 and AtClpC2 are more than 90% identical, though the extent of functional overlap between the two is not clear. Moreover, in vitro characterization reports are available only for AtClpC2, as AtClpC1 could not be expressed in recombinant form in the past. Herein, using a bacterial expression system, we have successfully expressed and purified AtClpC1 with a short N-terminal truncation, employing a three-step chromatographic purification strategy. We show that AtClpC1 exists as a hexamer in the presence of ATP and MgCl2, as known for other functional Clp chaperones. Further, our SAXS analyses provide a low-resolution envelope structure for the hexameric AtClpC1, which very well fits a ClpC hexamer model.

Analysis of Indian SARS-CoV-2 Genomes Reveals Prevalence of D614G Mutation in Spike Protein Predicting an Increase in Interaction With TMPRSS2 and Virus Infectivity.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has emerged as a global pandemic worldwide. In this study, we used ARTIC primers-based amplicon sequencing to profile 225 SARS-CoV-2 genomes from India. Phylogenetic analysis of 202 high-quality assemblies identified the presence of all the five reported clades 19A, 19B, 20A, 20B, and 20C in the population. The analyses revealed Europe and Southeast Asia as two major routes for introduction of the disease in India followed by local transmission. Interestingly, the19B clade was found to be more prevalent in our sequenced genomes (17%) compared to other genomes reported so far from India. Haplotype network analysis showed evolution of 19A and 19B clades in parallel from predominantly Gujarat state in India, suggesting it to be one of the major routes of disease transmission in India during the months of March and April, whereas 20B and 20C appeared to evolve from 20A. At the same time, 20A and 20B clades depicted prevalence of four common mutations 241 C > T in 5' UTR, P4715L, F942F along with D614G in the Spike protein. D614G mutation has been reported to increase virus shedding and infectivity. Our molecular modeling and docking analysis identified that D614G mutation resulted in enhanced affinity of Spike S1-S2 hinge region with TMPRSS2 protease, possibly the reason for increased shedding of S1 domain in G614 as compared to D614. Moreover, we also observed an increased concordance of G614 mutation with the viral load, as evident from decreased Ct value of Spike and the ORF1ab gene.

Structural Basis of Nucleosome Recognition and Modulation.

Chromatin structure and dynamics regulate key cellular processes such as DNA replication, transcription, repair, remodeling, and gene expression, wherein different protein factors interact with the nucleosomes. In these events, DNA and RNA polymerases, chromatin remodeling enzymes and transcription factors interact with nucleosomes, either in a DNA-sequence-specific manner and/or by recognizing different structural features on the nucleosome. The molecular details of the recognition of a nucleosome by different viral proteins, remodeling enzymes, histone post-translational modifiers, and RNA polymerase II, have been explored in the recent past. The present review puts forth critical insights into the basic mechanisms of nucleosome recognition by the various protein factors and the role of distinct surface epitopes on a nucleosome. These determinants of the underlying specificity include features such as the acidic patch, arginine anchor, histone post-translational modifications, core DNA, DNA lesions, and linker DNA.

AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains.

FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.

Structure-function relationship of H2A-H2B specific plant histone chaperones.

Studies on chromatin structure and function have gained a revived popularity. Histone chaperones are significant players in chromatin organization. They play a significant role in vital nuclear functions like transcription, DNA replication, DNA repair, DNA recombination, and epigenetic regulation, primarily by aiding processes such as histone shuttling and nucleosome assembly/disassembly. Like the other eukaryotes, plants also have a highly orchestrated and dynamic chromatin organization. Plants seem to have more isoforms within the same family of histone chaperones, as compared with other organisms. As some of these are specific to plants, they must have evolved to perform functions unique to plants. However, it appears that only little effort has gone into understanding the structural features of plant histone chaperones and their structure-function relationships. Studies on plant histone chaperones are essential for understanding their role in plant chromatin organization and how plants respond during stress conditions. This review is on the structural and functional aspects of plant histone chaperone families, specifically those which bind to H2A-H2B, viz nucleosome assembly protein (NAP), nucleoplasmin (NPM), and facilitates chromatin transcription (FACT). Here, we also present comparative analyses of these plant histone chaperones with available histone chaperone structures. The review hopes to incite interest among researchers to pursue further research in the area of plant chromatin and the associated histone chaperones.