The extremophile Picrophilus torridus carries a DNA adenine methylase M.PtoI that is part of a Type I restriction-modification system.

DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea. Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.

Correction: DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival.

[This corrects the article DOI: 10.1371/journal.ppat.1008190.].

ProSeqAProDB: Prosequence Assisted Protein Database.

In early 1990s, several proteins were shown to depend on additional stretches of polypeptide (termed as prosequence/prodomain) for their folding. These regions of the protein were often termed as IMCs (Intra Molecular Chaperones), since they would be cleaved from the mature folded protein eventually. Such proteins were hypothesized to face a kinetic barrier to their folding, which was probably lowered by the prosequences. In last three decades, numerous examples of such proteins have accumulated in literature. Yet, no study has been reported so far attempting to understand the evolutionary differences and similaritess of such proteins. Till date such proteins are continued to be treated as anomalous variants, rather than as representatives of any alternate protein folding strategy. Do such proteins have any distinctive structural facets OR typical biological roles, necessitating an unconventional strategy of protein folding? Do prosequences carry any unique or conserved features that are essential to their function? ProSeqAProDb: ProSequence Assisted Protein Database, (which can be accessed at https://proseqaprodb.mkulab.in) was built as a comprehensive database, to systematically study such proteins along with their pro-sequences. The database currently contains 2140 prosequence assisted proteins (1848 eukaryotic, 255 bacterial, 24 viral and 13 archaeal proteins), from 690 organisms later categorised into 960 families. We envisage that the availability of this curated dataset will enable the researchers worldwide to further their investigation in the origin, importance and evolution of such proteins, leading to better understanding of the protein folding process as a whole.

Potential of TCR sequencing in graft-versus-host disease.

Graft-versus-host disease (GvHD) remains one of the major complications following allogeneic haematopoietic stem cell transplantation (allo-HSCT). GvHD can occur in almost every tissue, with the skin, liver, and intestines being the mainly affected organs. T cells are implicated in initiating GvHD. T cells identify a broad range of antigens and mediate the immune response through receptors on their surfaces (T cell receptors, TCRs). The composition of TCRs within a T cell population defines the TCR repertoire of an individual, and this repertoire represents exposure to self and non-self proteins. Monitoring the changes in the TCR repertoire using TCR sequencing can provide an indication of the dynamics of a T cell population. Monitoring the frequency and specificities of specific TCR clonotypes longitudinally in different conditions and specimens (peripheral blood, GvHD-affected tissue samples) can provide insights into factors modulating immune reactions following allogeneic transplantation and will help to understand the underlying mechanisms mediating GvHD. This review provides insights into current studies of the TCR repertoire in GvHD and potential future clinical implications of TCR sequencing.

Insights into the Distribution and Functional Properties of l-Asparaginase in the Archaeal Domain and Characterization of Picrophilus torridus Asparaginase Belonging to the Novel Family Asp2like1.

l-Asparaginase catalyzes the hydrolysis of l-asparagine to aspartic acid and ammonia and is used in the medical and food industries. In this investigation, from the proteomes of 176 archaeal organisms (with completely sequenced genomes), 116 homologs of l-asparaginase were obtained from 86 archaeal organisms segregated into Asp1, Asp2, IaaA, Asp2like1, and Asp2like2 families based on the conserved domain. The similarities and differences in the structure of selected representatives from each family are discussed. From the two novel archaeal l-asparaginase families Asp2like1 and Asp2like2, a representative of Asp2like1 family Picrophilus torridus asparaginase (PtAsp2like1) was characterized in detail to find its suitability in therapeutics. PtAsp2like1 was a glutaminase-free asparaginase that showed the optimum activity at 80 °C and pH 10.0. The Km of PtAsp2like1 toward substrate l-asparagine was 11.69 mM. This study demonstrates the improved mapping of asparaginases in the archaeal domain, facilitating future focused research on archaeal asparaginases for therapeutic applications.

Mobile wallet adoption intention amid COVID-19 pandemic outbreak: A novel conceptual framework.

This research aims to explore adoption intention towards mobile wallet (m-wallet) amid COVID-19 outbreak using mediated-moderation framework. This study, in its uniqueness, utilises the stimulus-organism-response (S-O-R) theory as its theoretical base. The study investigated the effect of relative advantage, ease of effort, favourable infrastructure conditions, security considerations, and touch-free transactions on m-wallet adoption. The model includes perceived values as a mediator and perceived critical mass (PCM), promotional benefits (PBs) and users' demographics as moderators for deeper understanding of the phenomenon. A total of 327 responses were collected using purposive sampling method. The results revealed that relative advantage, favourable infrastructure conditions, security considerations and touch-free transactions exert a positive significant effect on m-wallet adoption intention. Further, except for ease of effort, perceived values mediate the association among antecedents and adoption intention and PCM, PBs and age found to be crucial moderators between perceived values and intention. This study enriches the existing literature on the adoption of m-wallet. Practically, this study helps marketers frame strategies to enhance the adoption and usage of m-wallet.

Active APPL1 sequestration by Plasmodium favors liver-stage development.

Intracellular pathogens manipulate host cells to survive and thrive. Cellular sensing and signaling pathways are among the key host machineries deregulated to favor infection. In this study, we show that liver-stage Plasmodium parasites compete with the host to sequester a host endosomal-adaptor protein (APPL1) known to regulate signaling in response to endocytosis. The enrichment of APPL1 at the parasitophorous vacuole membrane (PVM) involves an atypical Plasmodium Rab5 isoform (Rab5b). Depletion of host APPL1 alters neither the infection nor parasite development; however, upon overexpression of a GTPase-deficient host Rab5 mutant (hRab5_Q79L), the parasites are smaller and their PVM is stripped of APPL1. Infection with the GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) in this case rescues the PVM APPL1 signal and parasite size. In summary, we observe a robust correlation between the level of APPL1 retention at the PVM and parasite size during exoerythrocytic development.

Characterization of Cyclophilin from Thaumarchaeota Nitrosopumilus maritimus: Implications on the Diversity of Chaperone-like Activity in the Archaeal Domain.

The Archaea constitute separate domain of life and show resemblance with bacteria in their metabolic pathways while showing similarity with eukaryotes at the level of molecular processes such as cell division, DNA replication, protein synthesis, and proteostasis. However, the molecular machinery of archaea can be considered a simpler version of that found in eukaryotes because of the absence of multiple paralogs for any given molecular factor. Therefore, archaeal systems can possibly be used as a model system for understanding the eukaryotic protein folding machinery and thereby may help to address the molecular mechanism of various protein (mis)foldings and diseases. In the process of protein folding, the cis-trans isomerization of the peptide-prolyl bond is a rate-limiting step for the correct folding of proteins. Different types of peptidyl-prolyl cis-trans isomerases can accelerate this reaction, e.g., cyclophilin, FKBP, and parvulin. Among the five phyla of the archaeal domain, homologs of the cyclophilin protein are found only in two. Here we have characterized a cyclophilin from an archaeal organism, Nitrosopumilus maritimus (NmCyp), belonging to the phylum Thaumarchaeota. Like other known cyclophilins, NmCyp also possesses PPIase activity that can be inhibited by cyclosporine A. Generally, archaeal proteins are expected to possess differential thermal stability due to their adaptation to extreme environmental niche conditions. However, NmCyp exhibits low thermal stability and starts to aggregate beyond 40 °C. The properties of NmCyp are compared to those reported for the cyclophilin from another archaeal organism, Methanobrevibacter ruminantium. The current study sheds light on the differential behavior of cyclophilin proteins from two different phyla of archaea.

Distribution of Peptidyl-Prolyl Isomerase (PPIase) in the Archaea.

Cis-trans isomerization of the peptide bond prior to proline is an intrinsically slow process but plays an essential role in protein folding. In vivo cis-trans isomerization reaction is catalyzed by Peptidyl-prolyl isomerase (PPIases), a category of proteins widely distributed among all the three domains of life. The present study is majorly focused on the distribution of different types of PPIases in the archaeal domain. All the three hitherto known families of PPIases (namely FKBP, Cyclophilin and parvulin) were studied to identify the evolutionary conservation across the phylum archaea. The basic function of cyclophilin, FKBP and parvulin has been conserved whereas the sequence alignment suggested variations in each clade. The conserved residues within the predicted motif of each family are unique. The available protein structures of different PPIase across various domains were aligned to ascertain the structural variation in the catalytic site. The structural alignment of native PPIase proteins among various groups suggested that the apo-protein may have variable conformations but when bound to their specific inhibitors, they attain similar active site configuration. This is the first study of its kind which explores the distribution of archaeal PPIases, along with detailed structural and functional analysis of each type of PPIase found in archaea.

Efficacy of signal peptide predictors in identifying signal peptides in the experimental secretome of Picrophilous torridus, a thermoacidophilic archaeon.

Secretory proteins are important for microbial adaptation and survival in a particular environment. Till date, experimental secretomes have been reported for a few archaea. In this study, we have identified the experimental secretome of Picrophilous torridus and evaluated the efficacy of various signal peptide predictors (SPPs) in identifying signal peptides (SPs) in its experimental secretome. Liquid chromatography mass spectrometric (LC MS) analysis was performed for three independent P. torridus secretome samples and only those proteins which were common in the three experiments were selected for further analysis. Thus, 30 proteins were finally included in this study. Of these, 10 proteins were identified as hypothetical/uncharacterized proteins. Gene Ontology, KEGG and STRING analyses revealed that majority of the sercreted proteins and/or their interacting partners were involved in different metabolic pathways. Also, a few proteins like malate dehydrogenase (Q6L0C3) were multi-functional involved in different metabolic pathways like carbon metabolism, microbial metabolism in diverse environments, biosynthesis of antibiotics, etc. Multi-functionality of the secreted proteins reflects an important aspect of thermoacidophilic adaptation of P. torridus which has the smallest genome (1.5 Mbp) among nonparasitic aerobic microbes. SPPs like, PRED-SIGNAL, SignalP 5.0, PRED-TAT and LipoP 1.0 identified SPs in only a few secreted proteins. This suggests that either these SPPs were insufficient, or N-terminal SPs were absent in majority of the secreted proteins, or there might be alternative mechanisms of protein translocation in P. torridus.

Uncovering the structure-function aspects of an archaeal CsaA protein.

CsaA is known to function as a protein secretion chaperone in bacteria. Homologs of CsaA are also found in archaea while they are absent in eukaryotes. This paper presents the biophysical, biochemical analysis and crystallographic structure determination of CsaA from a thermoacidophilic archaeon Picrophilus torridus (PtCsaA). The PtCsaA appears to prevent the aggregation of heat denatured Bovine Carbonic Anhydrase II (BCAII). Differential denaturation of PtCsaA by guanidine hydrochloride (Gdn-HCl) and urea indicates the stabilization of the protein via salt bridges. Denaturant mediated decrease in 8-Anilinonaphthalene-1-sulfonic acid (ANS) binding and shift in wavelength signifies the partial unfolding of the protein molecule and exposure of hydrophobic patches to solvent on denaturation. The crystal structure of PtCsaA was solved to a resolution of 1.7 Å. The structure of PtCsaA appears to be similar to bacterial CsaA in architecture. Docking of a six amino acid peptide in the substrate binding pocket of PtCsaA suggests conservation in the substrate binding cavity. Residues involved in the formation of the binding cavity and hydrogen bonds responsible for the dimerization of PtCsaA were compared with those observed in the structure of Bacillus subtilis CsaA. The similarities and differences in electrostatic surface potential of the substrate binding cavities in bacterial CsaA and PtCsaA are discussed.

Identification of Binding Partners of CsaA - An Archaeal Chaperonic Protein of Picrophilus torridus.

BACKGROUND: CsaA is among the few chaperones which are present in both bacteria and archaea, but absent in eukaryotes. There are no reports on interactome analysis of CsaA from archaea, till date. Identification of binding partners of CsaA might be helpful in understanding CsaA-associated processes in Picrophilus torridus an extreme thermoacidophilic euryarchaeon. OBJECTIVES: The present study was conducted to identify the binding partners of CsaA of P. torridus (PtCsaA). METHODS: The binding partners of PtCsaA were isolated and identified using a pull down assay and liquid chromatography-mass spectrometry (LC-MS). RESULTS: The results revealed twelve potential binding partners of CsaA. These were thermosome subunits (Q6KZS2 and Q6L132), nascent polypeptide-associated complex protein (Q6L1N3), elongation factor 1-alpha (Q6L202), uncharacterized protein (Q6L0Y6), citrate synthase (Q6L0M8), asparaginyl- tRNA synthetase (Q6L0M5), succinyl-CoA synthetase beta chain (Q6L0B4), pyruvate ferredoxin oxidoreductase alpha and beta chain proteins (Q6KZA7 and Q6KZA6, respectively), malate dehydrogenase (Q6L0C3) and reversed fumarylacetoacetase (Q6KZ97). Functional categorization revealed that of these, six proteins were involved in energy metabolic pathways, three were archaeal chaperones, two were involved in translation and one might be a transcription regulator. STRING-based analysis of the protein-protein interactions of the experimental interactome revealed strong interactions among them. CONCLUSION: PtCsaA might be a multifaceted protein which besides translation might also play important role in metabolic processes of P. torridus. However, further experiments investigating the binding partners of CsaA in other archaea are required for a better understanding of CsaA-associated processes in archaea.

Heterogeneity in the endocytic capacity of individual macrophage in a population determines its subsequent phagocytosis, infectivity and subcellular trafficking.

Phagocytosis is a complex cellular uptake process involving multiple distinct steps of cargo recognition, uptake, phagosome maturation and eventual phagolysosome resolution. Emerging literature shows that heterogeneity of phagocytosis at multiple steps at a single cell level influences the population outcome. However, the determinants of phagocytic heterogeneity are not clear. Here we show that the variance in the endocytic capacity of individual cells in a macrophage population determines subsequent phagocytic uptake and trafficking. Our results document the extensive heterogeneity in the endocytic uptake of individual macrophages, and show that cells with higher endocytic capacity preferentially phagocytose diverse cargo, including pathogenic Mycobacterium tuberculosis. Interestingly, M. tuberculosis infected cells sustain the higher endocytic capacity following infection. Modulating endocytic capacity by inhibiting endocytosis reduces phagocytic uptake. Differential uptake of M. tuberculosis into cells with different endocytic capacities correlates with the efficiency of phagocytic delivery to lysosomes, thus contributing further to phagocytic as well as mycobacterial heterogeneity. Thus, variance in endocytic capacity is a determinant of generating heterogeneity in phagocytosis at multiple steps.

Biophysical and Biochemical Characterization of Nascent Polypeptide-Associated Complex of Picrophilus torridus and Elucidation of Its Interacting Partners.

Nascent polypeptide-associated complex (NAC) is a ribosome-associated molecular chaperone which is present only in archaea and eukaryotes. The primary function of NAC is to shield the newly synthesized polypeptide chains from inappropriate interactions with the cytosolic factors. Besides that, NAC has been implicated in diverse biological functions, which suggest that it might be a multifunctional protein. An elaborate study on NAC can provide useful information on protein folding in extreme conditions in which many archaea grow. Thus, in the present study, we have studied the biophysical and the biochemical characteristics of NAC of Picrophilus torridus, an extreme thermoacidophilic archaeon. The study of protein-protein interactions and binding partners of a protein provides useful insights into the new/unreported roles of a protein. Thus, in this study, we have identified the binding partners of NAC in P. torridus. The NAC protein of P. torridus was cloned, expressed, and purified, and its binding partners were isolated by a pull down assay followed by identification with liquid chromatography-mass spectrometry. To the best of our knowledge, this is the first report on the biophysical and the biochemical characterization of NAC from P. toridus and the identification of its interacting partners.

Mycobacterium tuberculosis (Mtb) lipid mediated lysosomal rewiring in infected macrophages modulates intracellular Mtb trafficking and survival.

Intracellular pathogens commonly manipulate the host lysosomal system for their survival. However, whether this pathogen-induced alteration affects the organization and functioning of the lysosomal system itself is not known. Here, using in vitro and in vivo infections and quantitative image analysis, we show that the lysosomal content and activity are globally elevated in Mycobacterium tuberculosis (Mtb)-infected macrophages. We observed that this enhanced lysosomal state is sustained over time and defines an adaptive homeostasis in the infected macrophage. Lysosomal alterations are caused by mycobacterial surface components, notably the cell wall-associated lipid sulfolipid-1 (SL-1), which functions through the mTOR complex 1 (mTORC1)-transcription factor EB (TFEB) axis in the host cells. An Mtb mutant lacking SL-1, MtbΔpks2, shows attenuated lysosomal rewiring compared with the WT Mtb in both in vitro and in vivo infections. Exposing macrophages to purified SL-1 enhanced the trafficking of phagocytic cargo to lysosomes. Correspondingly, MtbΔpks2 exhibited a further reduction in lysosomal delivery compared with the WT. Reduced trafficking of this mutant Mtb strain to lysosomes correlated with enhanced intracellular bacterial survival. Our results reveal that global alteration of the host lysosomal system is a defining feature of Mtb-infected macrophages and suggest that this altered lysosomal state protects host cell integrity and contributes to the containment of the pathogen.

DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival.

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids.

Biophysical and biochemical characterization of a thermostable archaeal cyclophilin from Methanobrevibacter ruminantium.

The archaeal protein folding machinery is quite similar to that found in eukaryotes, especially in terms of shared components like chaperones. Cyclophilins are chaperones found in both eukaryotes and archaea, which catalyze the reversible cis-trans isomerization around peptidyl-prolyl imide bond (PPIase activity). Eukaryotes possess multiple cyclophilin genes, many of which have acquired divergent functions. Archaea, having a single copy of this gene, may help better in comprehending the role of cyclophilins in maintaining cellular proteostasis. However, no cyclophilin homologs from archaea have been characterized as yet, limiting comparison with their eukaryotic counterparts. In the present work, we characterize in detail a cyclophilin from the archaea, Methanobrevibacter ruminantium (MrCyp). We explore the functional and structural characteristics of MrCyp using various biophysical techniques. MrCyp exhibits both the PPIase and aggregation prevention activity. Analysis of folding/unfolding data and measurement of ∆GNUH2O and Tm suggest that the protein is thermodynamically stable. MrCyp helps in increasing cell viability of E. coli cells. These features imply that MrCyp could be a promising candidate for co-expression mediated enhancement in the yield and quality of over-expressed proteins in heterologous expression systems such as E. coli. This is the first study of its kind, reporting the detailed functional characterization of an archaeal cyclophilin.

Insights into archaeal chaperone machinery: a network-based approach.

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.