Identification of functional pathways and potential genes associated with interferon signaling during human adenovirus type 7 infection by weighted gene coexpression network analysis.

Human adenovirus type 7 (HAdV-7) can cause severe pneumonia and complications in children. However, the mechanism of pathogenesis and the genes involved remain largely unknown. We collected HAdV-7-infected and mock-infected A549 cells at 24, 48, and 72 hours postinfection (hpi) for RNA sequencing (RNA-Seq) and identified potential genes and functional pathways associated with HAdV-7 infection using weighted gene coexpression network analysis (WGCNA). Based on bioinformatics analysis, 12 coexpression modules were constructed by WGCNA, with the blue, tan, and brown modules significantly positively correlated with adenovirus infection at 24, 48, and 72 hpi, respectively. Functional enrichment analysis indicated that the blue module was mainly enriched in DNA replication and viral processes, the tan module was largely enriched in metabolic pathways and regulation of superoxide radical removal, and the brown module was predominantly enriched in regulation of cell death. qPCR was used to determine transcript abundance of some identified hub genes, and the results were consistent with those from RNA-Seq. Comprehensively analyzing hub genes and differentially expressed genes in the GSE68004 dataset, we identified SOCS3, OASL, ISG15, and IFIT1 as potential candidate genes for use as biomarkers or drug targets in HAdV-7 infection. We propose a multi-target inhibition of the interferon signaling mechanism to explain the association of HAdV-7 infection with the severity of clinical consequences. This study has allowed us to construct a framework of coexpression gene modules in A549 cells infected with HAdV-7, thus providing a basis for identifying potential genes and pathways involved in adenovirus infection and for investigating the pathogenesis of adenovirus-associated diseases.

Localized islet nuclear enlargement hyperinsulinism (LINE-HI) due to ABCC8 and GCK mosaic mutations.

Objective: Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. Design: The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. Methods: We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Results: Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. Conclusion: These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases.

Vascular KATP channel structural dynamics reveal regulatory mechanism by Mg-nucleotides.

Vascular tone is dependent on smooth muscle KATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantú syndrome. Unique among KATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined cryogenic electron microscopy structures of vascular KATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic KATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational "propeller" and "quatrefoil" geometries surrounding their Kir6.1 core. The glutamate/aspartate-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. Molecular dynamics simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B, and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated, and MgADP-bound activated conformations wherein the glutamate/aspartate-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward KATP channel activation.

AKAP79/150 coordinates leptin-induced PKA signaling to regulate KATP channel trafficking in pancreatic ß-cells.

The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic ß-cell, which secretes insulin upon glucose stimulation. Leptin is known to suppress glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the ß-cell surface, which increases K+ conductance and causes ß-cell hyperpolarization. We have previously shown that leptin-induced KATP channel trafficking requires protein kinase A (PKA)-dependent actin remodeling. However, whether PKA is a downstream effector of leptin signaling or PKA plays a permissive role is unknown. Using FRET-based reporters of PKA activity, we show that leptin increases PKA activity at the cell membrane and that this effect is dependent on N-methyl-D-aspartate receptors, CaMKKß, and AMPK, which are known to be involved in the leptin signaling pathway. Genetic knockdown and rescue experiments reveal that the increased PKA activity upon leptin stimulation requires the membrane-targeted PKA-anchoring protein AKAP79/150, indicating that PKA activated by leptin is anchored to AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, leads to increased surface KATP channels even in the absence of leptin stimulation. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate KATP channel trafficking in ß-cells, hence insulin secretion. The study further advances our knowledge of the downstream signaling events that may be targeted to restore insulin secretion regulation in ß-cells defective in leptin signaling, such as those from obese individuals with type 2 diabetes.

CFTR trafficking mutations disrupt cotranslational protein folding by targeting biosynthetic intermediates.

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.

Mechanism of pharmacochaperoning in a mammalian KATP channel revealed by cryo-EM.

ATP-sensitive potassium (KATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic ß-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse KATP inhibitors are known to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of a mammalian KATP channel bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. We found all three drugs bind within a common pocket in SUR1. Further, we found the N-terminus of Kir6.2 inserted within the central cavity of the SUR1 ABC core, adjacent the drug binding pocket. The findings reveal a common mechanism by which diverse compounds stabilize the Kir6.2 N-terminus within SUR1's ABC core, allowing it to act as a firm 'handle' for the assembly of metastable mutant SUR1-Kir6.2 complexes.

The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.

Protein folding. Translational tuning optimizes nascent protein folding in cells.

In cells, biosynthetic machinery coordinates protein synthesis and folding to optimize efficiency and minimize off-pathway outcomes. However, it has been difficult to delineate experimentally the mechanisms responsible. Using fluorescence resonance energy transfer, we studied cotranslational folding of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator. During synthesis, folding occurred discretely via sequential compaction of N-terminal, α-helical, and α/ß-core subdomains. Moreover, the timing of these events was critical; premature α-subdomain folding prevented subsequent core formation. This process was facilitated by modulating intrinsic folding propensity in three distinct ways: delaying α-subdomain compaction, facilitating ß-strand intercalation, and optimizing translation kinetics via codon usage. Thus, de novo folding is translationally tuned by an integrated cellular response that shapes the cotranslational folding landscape at critical stages of synthesis.

Cotranslational stabilization of Sec62/63 within the ER Sec61 translocon is controlled by distinct substrate-driven translocation events.

The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αßγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62, and Sec63. Moreover, Sec62/63 stabilization within the translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. These data show that cotranslational translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.

Kinetic analysis of ribosome-bound fluorescent proteins reveals an early, stable, cotranslational folding intermediate.

Protein folding in cells reflects a delicate interplay between biophysical properties of the nascent polypeptide, the vectorial nature and rate of translation, molecular crowding, and cellular biosynthetic machinery. To better understand how this complex environment affects de novo folding pathways as they occur in the cell, we expressed ß-barrel fluorescent proteins derived from GFP and RFP in an in vitro system that allows direct analysis of cotranslational folding intermediates. Quantitative analysis of ribosome-bound eCFP and mCherry fusion proteins revealed that productive folding exhibits a sharp threshold as the length of polypeptide from the C terminus to the ribosome peptidyltransferase center is increased. Fluorescence spectroscopy, urea denaturation, and limited protease digestion confirmed that sequestration of only 10-15 C-terminal residues within the ribosome exit tunnel effectively prevents stable barrel formation, whereas folding occurs unimpeded when the C terminus is extended beyond the ribosome exit site. Nascent FPs with 10 of the 11 ß-strands outside the ribosome exit tunnel acquire a non-native conformation that is remarkably stable in diverse environments. Upon ribosome release, these structural intermediates fold efficiently with kinetics that are unaffected by the cytosolic crowding or cellular chaperones. Our results indicate that during synthesis, fluorescent protein folding is initiated cotranslationally via rapid formation of a highly stable, on-pathway structural intermediate and that the rate-limiting step of folding involves autonomous incorporation of the 11th ß-strand into the mature barrel structure.

Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex.

In eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility, and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. Importantly, type II SA inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment.

Ligand-driven vectorial folding of ribosome-bound human CFTR NBD1.

The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (FRET) to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389-500 emerge from the ribosome exit tunnel, initiating compaction of a small, N-terminal α/ß-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

In vitro incorporation of nonnatural amino acids into protein using tRNA(Cys)-derived opal, ochre, and amber suppressor tRNAs.

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA(Cys) that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.

Sequence-specific retention and regulated integration of a nascent membrane protein by the endoplasmic reticulum Sec61 translocon.

A defining feature of eukaryotic polytopic protein biogenesis involves integration, folding, and packing of hydrophobic transmembrane (TM) segments into the apolar environment of the lipid bilayer. In the endoplasmic reticulum, this process is facilitated by the Sec61 translocon. Here, we use a photocross-linking approach to examine integration intermediates derived from the ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) and show that the timing of translocon-mediated integration can be regulated at specific stages of synthesis. During CFTR biogenesis, the eighth TM segment exits the ribosome and enters the translocon in proximity to Sec61alpha. This interaction is initially weak, and TM8 spontaneously dissociates from the translocon when the nascent chain is released from the ribosome. Polypeptide extension by only a few residues, however, results in stable TM8-Sec61alpha photocross-links that persist after peptidyl-tRNA bond cleavage. Retention of these untethered polypeptides within the translocon requires ribosome binding and is mediated by an acidic residue, Asp924, near the center of the putative TM8 helix. Remarkably, at this stage of synthesis, nascent chain release from the translocon is also strongly inhibited by ATP depletion. These findings contrast with passive partitioning models and indicate that Sec61alpha can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.