Nutrient Signaling-Dependent Quaternary Structure Remodeling Drives the Catalytic Activation of metazoan PASK.

The Per-Arnt-Sim (PAS) domains are characterized by diverse sequences and feature tandemly arranged PAS and PAS-associated C-terminal (PAC) motifs that fold seamlessly to generate the metabolite-sensing PAS domain. Here, using evolutionary scale sequence, domain mapping, and deep learning-based protein structure analysis, we deconstructed the sequence-structure relationship to unearth a novel example of signal-regulated assembly of PAS and PAC subdomains in metazoan PAS domain-regulated kinase (PASK). By comparing protein sequence, domain architecture, and computational protein models between fish, bird, and mammalian PASK orthologs, we propose the existence of previously unrecognized third PAS domain of PASK (PAS-C) formed through long-range intramolecular interactions between the N-terminal PAS fold and the C-terminal PAC fold. We experimentally validated this novel structural design using residue-level cross-linking assays and showed that the PAS-C domain assembly is nutrient-responsive. Furthermore, by combining structural phylogeny approaches with residue-level cross-linking, we revealed that the PAS-C domain assembly links nutrient sensing with quaternary structure reorganization in PASK, stabilizing the kinase catalytic core of PASK. Thus, PAS-C domain assembly likely integrates environmental signals, thereby relaying sensory information for catalytic control of the PASK kinase domain. In conclusion, we theorize that during their horizontal transfer from bacteria to multicellular organisms, PAS domains gained the capacity to integrate environmental signals through dynamic modulation of PAS and PAC motif interaction, adding a new regulatory layer suited for multicellular systems. We propose that metazoan PAS domains are likely to be more dynamic in integrating sensory information than previously considered, and their structural assembly could be targeted by regulatory signals and exploited to develop therapeutic strategies.

Signal-regulated Unmasking of Nuclear Localization Motif in the PAS Domain Regulates the Nuclear Translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells through intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. This interaction serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting this association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the intramolecular interaction in PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

Signal-regulated unmasking of the nuclear localization motif in the PAS domain regulates the nuclear translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells via intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. The interaction between the PAS-A domain and PIM is evolutionarily conserved and serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting the PAS-A: PIM association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the PAS-A domain of PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence.

Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence, Antimicrobial Susceptibility Pattern, and Detection of mecA Gene among Cardiac Patients from a Tertiary Care Heart Center in Kathmandu, Nepal.

BACKGROUND: Methicillin Resistant Staphylococcus aureus (MRSA) is a significant human pathogen associated with nosocomial infections. mecA in the S. aureus is a marker of MRSA. The main objective of this study was to detect mecA and vanA genes conferring resistance in S. aureus among cardiac patients attending Sahid Gangalal National Heart Centre (SGNHC), Kathmandu, Nepal between May and November 2019. METHODS: A total of 524 clinical samples (blood, urine, sputum) were collected and processed. Bacterial isolates were tested for antimicrobial susceptibility test (AST) and screening for MRSA was carried out by cefoxitin disc diffusion method. Minimum inhibitory concentration (MIC) of vancomycin for MRSA was established by agar dilution method and chromosomal DNA was extracted and used in polymerase chain reaction targeting the mecA and vanA genes. RESULTS: Out of 524 specimens, 27.5% (144/524) showed bacterial growth. Among 144 culture positive isolates, S. aureus (27.1%; 39/144) was the predominant bacteria. Among 39 S. aureus isolates, all isolates were found resistant to penicillin followed by erythromycin (94.9%; 37/39), gentamicin (94.9%; 37/39) and cefoxitin (87.2%; 34/39). Out of 39 S. aureus, 87.2% (34/39) were MRSA. Among 34 MRSA, 8.8% (3/34) were vancomycin intermediate S. aureus (VISA). None of the MRSA was resistant to vancomycin. All of the 3 VISA isolates were obtained from inpatients. Of 39 S. aureus, 82.1% (32/39) harbored mecA gene. Similarly, the entire VISA isolates and 94.1% (32/34) of the MRSA isolates were tested positive for mecA gene. CONCLUSIONS: High prevalence of MRSA among the cardiac patients indicates the increasing burden of drug resistance among bacterial isolates. Since infection control is the crucial step in coping with the burgeoning antimicrobial resistance in the country, augmentation of diagnostic facilities with routine monitoring of drug resistance is recommended.