Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in ...many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.
Abstract
MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs). The assembly process establishes the seed, ...central, 3′ supplementary, and tail regions across the loaded guide, enabling the RISC to recognize target RNAs for silencing. This guide segmentation is caused by anchoring the 3′ end at the AGO PAZ domain, but the minimum guide length required for the conformation remains to be studied because the current miRNA size defined by Dicer processing is ambiguous. Using a 3′ → 5′ exonuclease ISG20, we determined the lengths of AGO-associated miR-20a and let-7a with 3′ ends that no longer reach the PAZ domain. Unexpectedly, miR-20a and let-7a needed different lengths, 19 and 20 nt, respectively, to maintain their RISC conformation. This difference can be explained by the low affinity of the PAZ domain for the adenosine at g19 of let-7a, suggesting that the tail-region sequence slightly alters the minimum guide length. We also present that 17-nt guides are sufficiently short enough to function as tinyRNAs (tyRNAs) whose 3′ ends are not anchored at the PAZ domain. Since tyRNAs do not have the prerequisite anchoring for the standardized guide segmentation, they would recognize targets differently from miRNAs and siRNAs.
The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for ...degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.
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•Hsc70/Hsp70 engage in novel bipartite binding mode with CHIP•Hsp70-lid interaction with CHIP is required for ubiquitination of Hsp70 clients•TPR:lid-tail structure allows modeling of full-length Hsp70:CHIP complexes•Phosphorylation or methylation of Hsp70-lid residues regulate interaction with CHIP
Zhang et al. report a novel interaction between Hsp70 and CHIP. This interaction allows for full-length models of the CHIP/Hsp70 complex to be assembled, predicts how Hsp70 posttranslational modifications regulate ubiquitination, and suggests a new mode of chaperone/cochaperone interactions.
Heat-shock protein 90 (Hsp90) is one of the most important chaperones involved in multiple cellular processes. The chaperoning function of Hsp90 is intimately coupled to the ATPase activity presented ...by its N-terminal domain. However, the molecular mechanism for the ATP-dependent working cycle of Hsp90 is still not fully understood. In this study, we use NMR techniques to investigate the structural characteristics and dynamic behaviors of Hsp90 N-terminal domain in its free and AMPPCP (ATP analogue) or ADP-bound states. We demonstrated that although AMPPCP and ADP bind to almost the same region of Hsp90, significantly different effects on the dynamics behaviors of the key structural elements were observed. AMPPCP binding favors the formation of the active homodimer of Hsp90 by enhancing the slow-motion featured conformational exchanges of those residues (A117-A141) within the lid segment (A111-G135) and around region, while ADP binding keeps Hsp90 staying at the inactive state by increasing the conformational rigidity of the lid segment and around region. Based on our findings, a dynamic working model for the ATP-dependent functioning cycle of Hsp90 was proposed.
Methazolamide in high-altitude illnesses Lu, Hui; Zhang, Huaqun; Jiang, Yuanying
European journal of pharmaceutical sciences,
05/2020, Letnik:
148
Journal Article
Recenzirano
•As a carbonic anhydrase inhibitor, methazolamide improves ventilation and oxygenation level via inhibiting carbonic anhydrases.•Methazolamide can decrease oxidative stress by its non-carbonic ...anhydrase inhibitor functions.•Less fatigue side effects of methazolamide than acetazolamide.
As a carbonic anhydrase inhibitor and a methylated lipophilic analogue of acetazolamide, Methazolamide has higher lipid solubility, less plasma protein binding and renal excretion, and fewer side effects, compared to acetazolamide. Methazolamide can increase systemic metabolic acidosis and sequentially improve ventilation and oxygenation level. The increased oxygenation level leads to reduced reactive oxygen species (ROS) production, relived cerebral edema, mitigated hypoxic pulmonary vasoconstriction, abrogated hypoxic fatigue, and decreased excessive erythrocytosis. In addition to the effect as a carbonic anhydrase inhibitor, methazolamide directly activates the transcription factor anti-oxidative nuclear factor-related factor 2 (Nrf2) and inhibits interleukin-1β (IL-1β) release. These pharmacological functions of methazolamide are beneficial for the prevention and treatment of high-altitude illnesses. Besides, methazolamide causes less fatigue side effects than acetazolamide does. It is also worth noting that several studies suggested that a lower dose of methazolamide has similar prophylaxis and treatment efficacy in acute mountain sickness (AMS) to a higher dose of acetazolamide. Given methazolamide's advantages over acetazolamide, methazolamide may thus represent an alternative for acetazolamide when taken for high-altitude illnesses prophylaxis and treatment. However, more in-depth clinical trials are needed to fully evaluate this efficacy of methazolamide.
Heat shock protein (Hsp) 70 is the main mediator of protein quality control in the cytosol of eukaryotic cells. One of the major functions of Hsp70 is mediate the ubiquitination of client proteins ...which subsequently leads to proteasomal degradation. This process occurs via a multiprotein complex composed of Hsp70, carboxy terminus of Hsc70 interacting protein (CHIP), ubiquitin‐conjugating enzyme (E2), and ubiquitin (Ub). This complex is thought to be flexible, however, knowledge of the degree of dynamics are still lacking and detailed experimental evidence is needed. Here we present the models derived from Electron Paramagnetic Resonance (EPR) data and small‐angle X‐ray scattering (SAXS) ensembles for the complexes of Hsp70/CHIP/E2‐Ub with ATP or ADP. The model of ATP bound Hsp70/CHIP/E2‐Ub complex has an average distance of ~120 Å between the substrate‐binding domain (SBD) and the E2‐ubiquitin active site. The same distance of the multiprotein complex is found to be decreased to ~60 Å when Hsp70 is bound to ADP. Since the ADP bound conformation is considered as the active conformation that tightly binds client proteins, the alteration of the average distance between the Hsp70 SBD and the E2‐Ub active site implies that the Lys residue of the substrate might need to be positioned within appropriate distance to span the gap in order to efficiently promote ubiquitination. Here, we used molecular dynamic simulations, double electron‐electron resonance (DEER) EPR spectroscopy and in‐vitroubiquitination assays with designed model substrate proteins that place lysine residues at variable positions. We found the dynamics of the Hsp70/CHIP/E2‐Ubiquitin complex is playing a major role in the ubiquitination of various client proteins and these dynamics are necessary to overcome the distance barrier between E2‐Ub active site and the Hsp70 SBD. Our data also suggests that the Hsp70/CHIP complex is capable of ubiquitinating a client protein substrate when lysine residues are placed at several disparate positions. In sum, our data reveals the functional dynamics of the Hsp70/CHIP/E2‐Ub complex which enable ubiquitination of a client protein.
HIV-1 Tat is an important regulatory protein involved in AIDS pathogenesis. However, the immunoprofiles of anti-Tat responses remain unclear. We analysed the immunoprofiles of the anti-Tat antibody ...responses and the neutralizing activities. Out of 326 HIV-1-seropositive individuals, 12.9% were positive for anti-Tat antibodies. We found six different immunological profiles of anti-Tat antibody responses: full-potential response, combined response, N-specific response, C-specific response, full-length Tat-specific response and Tat-related response. These responses represent two types of anti-Tat responses: the major complete response and the alternative C-prone response. A Tat-neutralizing activity is significantly higher in anti-Tat-seropositive samples than anti-Tat-negative or healthy blood-donor samples, and significantly correlates with the anti-Tat reactivities. The data here could contribute to a better understanding of the significance of anti-Tat responses in preventing HIV pathogenesis and could be useful for designing more effective vaccines in the future.
Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of ...anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases.
The characterization of functional yet nonprotein coding (nc) RNAs has expanded the role of RNA in the cell from a passive player in the central dogma of molecular biology to an active regulator of ...gene expression. The misregulation of ncRNA function has been linked with a variety of diseases and disorders ranging from cancers to neurodegeneration. However, a detailed molecular understanding of how ncRNAs function has been limited; due, in part, to the difficulties associated with obtaining high‐resolution structures of large RNAs. Tertiary structure determination of RNA as a whole is hampered by various technical challenges, all of which are exacerbated as the size of the RNA increases. Namely, RNAs tend to be highly flexible and dynamic molecules, which are difficult to crystallize. Biomolecular nuclear magnetic resonance (NMR) spectroscopy offers a viable alternative to determining the structure of large RNA molecules that do not readily crystallize, but is itself hindered by some technical limitations. Recently, a series of advancements have allowed the biomolecular NMR field to overcome, at least in part, some of these limitations. These advances include improvements in sample preparation strategies as well as methodological improvements. Together, these innovations pave the way for the study of ever larger RNA molecules that have important biological function.
This article is categorized under:
RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
Overview of important sample preparation and methodological advancements that facilitate the study of large RNA structure and dynamics by nuclear magnetic resonance spectroscopy. These innovations pave the way for the study of previously intractable systems.
RNA thermosensors (RNATs), found in the 5' untranslated region (UTR) of some bacterial messenger RNAs (mRNAs), control the translation of the downstream gene in a temperature-dependent manner. In
, ...the expression of a key transcription factor, PrfA, is mediated by an RNAT in its 5' UTR. PrfA functions as a master regulator of virulence in
, controlling the expression of many virulence factors. The temperature-regulated expression of PrfA by its RNAT element serves as a signal of successful host invasion for the bacteria. Structurally, the prfA RNAT bears little resemblance to known families of RNATs, and prior studies demonstrated that the prfA RNAT is highly responsive over a narrow temperature range. Herein, we have undertaken a comprehensive mutational and thermodynamic analysis to ascertain the molecular determinants of temperature sensitivity. We provide evidence to support the idea that the prfA RNAT unfolding is different from that of cssA, a well-characterized RNAT, suggesting that these RNATs function via distinct mechanisms. Our data show that the unfolding of the prfA RNAT occurs in two distinct events and that the internal loops play an important role in mediating the cooperativity of RNAT unfolding. We further demonstrated that regions distal to the ribosome binding site (RBS) not only contribute to RNAT structural stability but also impact translation of the downstream message. Our collective results provide insight connecting the thermal stability of the prfA RNAT structure, unfolding energetics, and translational control.
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