The ABCF proteins in Escherichia coli individually cope with 'hard-to-translate' nascent peptide sequences.

Organisms possess a wide variety of proteins with diverse amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various problematic nascent peptide sequences within the exit tunnel. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that ATP hydrolysis-coupled structural rearrangement and the interdomain linker sequence are pivotal for handling 'hard-to-translate' nascent peptides. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences.

Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation.

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

Characteristics and outcomes of out-of-hospital cardiac arrest in a hilly area: Utstein Registry data from the Nagasaki Medical Region, Japan.

Aim: To analyze characteristics and investigate prognostic indicators of out-of-hospital cardiac arrest (OHCA) in a hilly area in Japan. Methods: A retrospective population-based study was conducted using the Utstein Registry for 4280 OHCA patients in the Nagasaki Medical Region (NMR) registered over the 10-year period from 2011 to 2020. The main outcome measure was a favorable cerebral performance category (CPC 1-2). Sites at which OHCA occurred were classified into "sloped places (SPs)" (not easily accessible by emergency medical services [EMS] personnel due to slopes) and "accessible places (APs)" (EMS personnel could park an ambulance close to the site). The characteristics and prognosis based on CPC were compared between SPs and APs, and multivariable analysis was performed. Results: No significant improvement in prognosis occurred in the NMR from 2011 to 2020. Prognosis in SPs was significantly worse than that in APs. However, multivariable analysis did not identify SP as a prognostic indicator. The following factors were associated with survival and CPC 1-2: age group, witness status, first documented rhythm, bystander-initiated cardiopulmonary resuscitation (CPR) and automated external defibrillator (AED) use, use of mechanical CPR (m-CPR) device or esophageal obturator airway (EOA), and year. Both m-CPR and EOA use were associated with a poor prognosis. Conclusion: In a hilly area, OHCA patients in SPs had a worse prognosis than those in APs, but SPs was not significantly associated with prognosis by multivariable analysis. Interventions to increase bystander-initiated CPR and AED use could potentially improve outcomes of OHCA in the NMR.

Prediction of chaperonin GroE substrates using small structural patterns of proteins.

Molecular chaperones are indispensable proteins that assist the folding of aggregation-prone proteins into their functional native states, thereby maintaining organized cellular systems. Two of the best-characterized chaperones are the Escherichia coli chaperonins GroEL and GroES (GroE), for which in vivo obligate substrates have been identified by proteome-wide experiments. These substrates comprise various proteins but exhibit remarkable structural features. They include a number of α/ß proteins, particularly those adopting the TIM ß/α barrel fold. This observation led us to speculate that GroE obligate substrates share a structural motif. Based on this hypothesis, we exhaustively compared substrate structures with the MICAN alignment tool, which detects common structural patterns while ignoring the connectivity or orientation of secondary structural elements. We selected four (or five) substructures with hydrophobic indices that were mostly included in substrates and excluded in others, and developed a GroE obligate substrate discriminator. The substructures are structurally similar and superimposable on the 2-layer 2α4ß sandwich, the most popular protein substructure, implying that targeting this structural pattern is a useful strategy for GroE to assist numerous proteins. Seventeen false positives predicted by our methods were experimentally examined using GroE-depleted cells, and 9 proteins were confirmed to be novel GroE obligate substrates. Together, these results demonstrate the utility of our common substructure hypothesis and prediction method.

The expression of repulsive guidance molecule a after traumatic brain injury: Time-course changes in gene expression in a murine model of controlled cortical impact.

INTRODUCTION: Repulsive guidance molecule a (RGMa) is a key protein that negatively regulates neuronal regeneration as its inhibition enhances axonal growth and promotes functional recovery in animal models of spinal cord injury. However, the role of RGMa in traumatic brain injury (TBI) remains elusive. This study aimed to clarify TBI-responsive RGMa expression in a murine model. METHODS: Adult male C57BL/6J mice were subjected to controlled cortical impact. Brains were extracted 6 hours and 1, 3, 7, 14 and 21 days after injury (n = 6 in each group). Changes in the messenger RNA (mRNA) expression of RGMa and its receptor, neogenin, were evaluated by quantitative polymerase chain reaction in the damaged area of the cortex and contralateral cortex, along with expression measurement of inflammation-related molecules. Neurological deficit was also assessed by the cylinder test. RESULTS: Neurological score was consistently lower in the TBI group compared to the sham group throughout the experimental period. The mRNA expressions of representative inflammatory cytokine TNF-α and chemokine receptor CCR2 were remarkably increased in the injured cortex on day 1 and gradually decreased over time, although remaining at higher values at least until day 14. The mRNA expressions of RGMa and neogenin were significantly suppressed in the damaged cortex until day 3. Interestingly, RGMa expression was suppressed most on day 1 and recovered over time. CONCLUSION: In the acute phase of TBI, gene expression of inflammatory cytokines significantly increased, and gene expressions of RGMa and neogenin significantly decreased in the inflammatory milieu of the damaged area. Despite the subsequent remission of inflammation, RGMa gene expression recovered to the normal level 1 week after TBI. Intrinsic regenerative response to acute brain injury might be hampered by the following recovery of RGMa expression, hinting at the possibility of functional RGMa inhibition as a new, effective maneuver against TBI.

Translation-coupled protein folding assay using a protease to monitor the folding status.

Protein folding is an essential prerequisite for proteins to execute nearly all cellular functions. There is a growing demand for a simple and robust method to investigate protein folding on a large-scale under the same conditions. We previously developed a global folding assay system, in which proteins translated using an Escherichia coli-based cell-free translation system are centrifuged to quantitate the supernatant fractions. Although the assay is based on the assumption that the supernatants contain the folded native states, the supernatants also include nonnative unstructured proteins. In general, proteases recognize and degrade unstructured proteins, and thus we used a protease to digest the unstructured regions to monitor the folding status. The addition of Lon protease during the translation of proteins unmasked subfractions, not only in the soluble fractions but also in the aggregation-prone fractions. We translated ∼90 E. coli proteins in the protease-inclusion assay, in the absence and presence of chaperones. The folding assay, which sheds light on the molecular mechanisms underlying the aggregate formation and the chaperone effects, can be applied to a large-scale analysis.

Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding.

A subset of the proteome is prone to aggregate formation, which is prevented by chaperones in the cell. To investigate whether the basic principle underlying the aggregation process is common in prokaryotes and eukaryotes, we conducted a large-scale aggregation analysis of ~500 cytosolic budding yeast proteins using a chaperone-free reconstituted translation system, and compared the obtained data with that of ~3,000 Escherichia coli proteins reported previously. Although the physicochemical properties affecting the aggregation propensity were generally similar in yeast and E. coli proteins, the susceptibility of aggregation in yeast proteins were positively correlated with the presence of intrinsically disordered regions (IDRs). Notably, the aggregation propensity was not significantly changed by a removal of IDRs in model IDR-containing proteins, suggesting that the properties of ordered regions in these proteins are the dominant factors for aggregate formation. We also found that the proteins with longer IDRs were disfavored by E. coli chaperonin GroEL/ES, whereas both bacterial and yeast Hsp70/40 chaperones have a strong aggregation-prevention effect even for proteins possessing IDRs. These results imply that a key determinant to discriminate the eukaryotic proteomes from the prokaryotic proteomes in terms of protein folding would be the attachment of IDRs.

Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system.

Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of >90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins.