Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation

Electron and photon reconstruction and identification with the CMS experiment at the CERN LHC

The performance is presented of the reconstruction and identification algorithms for electrons and photons with the CMS experiment at the LHC. The reported results are based on proton-proton collision data collected at a center-of-mass energy of 13 TeV and recorded in 2016-2018, corresponding to an integrated luminosity of 136 fb(-1). Results obtained from lead-lead collision data collected at root S-NN = 5.02 TeV are also presented. Innovative techniques are used to reconstruct the electron and photon signals in the detector and to optimize the energy resolution. Events with electrons and photons in the final state are used to measure the energy resolution and energy scale uncertainty in the recorded events. The measured energy resolution for electrons produced in Z boson decays in proton-proton collision data ranges from 2 to 5%, depending on electron pseudorapidity and energy loss through bremsstrahlung in the detector material. The energy scale in the same range of energies is measured with an uncertainty smaller than 0.1 (0.3)% in the barrel (endcap) region in proton-proton collisions and better than 1(3)% in the barrel (endcap) region in heavy ion collisions. The timing resolution for electrons from Z boson decays with the full 2016-2018 proton-proton collision data set is measured to be 200 ps.Peer reviewe

Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90 beta

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90 beta. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90 beta inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90 beta and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90 beta play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation

Molecular chaperone-mediated protein quality control of human CLC-1 chloride channel

先天性肌肉強直症 (myotonia congenita) 是一種遺傳性肌肉疾病，是由於電壓敏感性氯離子通道CLC-1基因發生突變所造成。A531V是一種先天性肌肉強直症的突變型，其開關特性 (gating property) 與野生型 (wild-type) 並無明顯差別；但A531V蛋白質的表現量卻明顯較少，此表現減少的原因之一是由於蛋白酶體降解增加。本實驗室先前的研究發現A531V與伴護蛋白Hsp90的輔助伴護蛋白Aha1以及帶有PPIase功能的伴護蛋白 FKBP8有交互作用。分子伴護蛋白可協助蛋白質的折疊，以及將不具功能、錯誤摺疊的蛋白經由降解途徑移除。本篇論文目的為檢測可能調控CLC-1的chaperon，並且了解 CLC-1 WT和A531V是否經由Hsp70-Hsp90伴護蛋白系統來調控其蛋白質的品質控管。 我們發現大量表現Aha1可增加CLC-1 WT與A531V的穩定性，而利用shRNA壓制內生性Aha1的表現則降低了CLC-1 WT與A531V的表現；這個現象顯示加速Hsp90 ATPase cycling的速率可協助CLC-1的摺疊。此外，我們觀察到大量表現FKBP8後同樣也可增加CLC-1 WT與A531V的穩定性，並且增加了CLC-1在細胞膜上的表現，同時還可減短A531V的半衰期。從上述結果暗示CLC-1很有可能會經由Hsp70-Hsp90進行品質控管，接著我們大量表現Hsc70以及Hsp90α/β，並發現Hsc70以及Hsp90β同樣的增加了CLC-1的穩定性，但Hsp90α則對CLC-1沒有影響。當我們壓制了內生性的Hsc70之後，發現CLC-1 A531V表現量下降。我們也使用了Hsp90的抑制劑17-AAG，並發現CLC-1 WT和A531V的表現皆上升，此現象可能與17-AAG活化了Hsf1並造成其他伴護蛋白增加有關。 從我們的研究結果推測， Hsc70-Hsp90-FKBP8摺疊路徑可能是調控CLC-1蛋白質平衡的關鍵步驟。對於與肌強直症相關的圖變A531V而言，或許經由調控這些相關伴護蛋白的表現可以達成改善其不穩定缺陷的效果。Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl-) channel CLC-1. A531V is a myotonia-related mutant with a gating property similar to that of wild-type (WT) channels. The protein expression of A531V, however, is significantly lower, which is partly attributed to an enhanced proteasomal degradation. Previous studies from our lab demonstrated that CLC-1 may interact with the Hsp90 cochaperone Aha1, as well as with the PPIase chaperone FKBP8. Molecular chaperones are known to assist protein folding and to remove nonfunctional, misfolded, or aggregated proteins via degradation pathways. In this study, we aim to identify CLC-1-associated molecular chaperones, and to examine whether the chaperones and cochaperones of the Hsp70-Hsp90 system may contribute to the protein quality control of CLC-1 WT and A531V. Overexpression of Aha1 promoted protein expression of both WT and A531V, whereas knockdown of Aha1 reduced the protein expression of both WT and A531V. These observations suggest that the rate of Hsp90 ATPase cycling may be important for the correct folding of CLC-1. Overexpression of FKBP8 also promoted total and surface protein expression of WT and A531V, although only A531V displayed a significant increase in protein half-life. We then investigated the role of Hsp70 and Hsp90 in CLC-1 quality control by overexpressing Hsc70, Hsp90α, or Hsp90β. We found that both Hsc70 and Hsp90β, but not Hsp90α, increased the protein expression of WT and A531V. In addition, knockdown of Hsc70 decreased the expression of A531V. Surprisingly, treatment with 17-AAG, an Hsp90 inhibitor, also led to an increase in the expression of both WT and A531V, which may result from 17-AAG –induced expression of heat shock transcription factor 1 (Hsf1). Together, our data suggest that the Hsc70-Hsp90β-FKBP8 chaperone pathway may play a key role in controlling the protein homeostasis of CLC-1. Our findings also imply that manipulation of the expression/activity of these chaperones/cochaperones may partially correct the protein expression deficit of the myotonia-related mutant A531V

FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

Mutations in the skeletal muscle-specific CLC-1 chloride channel are associated with the human hereditary disease myotonia congenita. The molecular pathophysiology underlying some of the disease-causing mutations can be ascribed to defective human CLC-1 protein biosynthesis. CLC-1 protein folding is assisted by several molecular chaperones and co-chaperones, including FK506-binding protein 8 (FKBP8). FKBP8 is generally considered an endoplasmic reticulum- and mitochondrion-resident membrane protein, but is not thought to contribute to protein quality control at the cell surface. Herein, we aim to test the hypothesis that FKBP8 may regulate CLC-1 protein at the plasma membrane. Surface biotinylation and subcellular fractionation analyses reveal that a portion of FKBP8 is present at the plasma membrane, and that co-expression with CLC-1 enhances surface localization of FKBP8. Immunoblotting analyses of plasma membrane proteins purified from skeletal muscle further confirm surface localization of FKBP8. Importantly, FKBP8 promotes CLC-1 protein stability at the plasma membrane. Together, our data underscore the importance of FKBP8 in the peripheral quality control of CLC-1 channel

The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels

Defective Gating and Proteostasis of Human ClC-1 Chloride Channel: Molecular Pathophysiology of Myotonia Congenita.

The voltage-dependent ClC-1 chloride channel, whose open probability increases with membrane potential depolarization, belongs to the superfamily of CLC channels/transporters. ClC-1 is almost exclusively expressed in skeletal muscles and is essential for stabilizing the excitability of muscle membranes. Elucidation of the molecular structures of human ClC-1 and several CLC homologs provides important insight to the gating and ion permeation mechanisms of this chloride channel. Mutations in the human CLCN1 gene, which encodes the ClC-1 channel, are associated with a hereditary skeletal muscle disease, myotonia congenita. Most disease-causing CLCN1 mutations lead to loss-of-function phenotypes in the ClC-1 channel and thus increase membrane excitability in skeletal muscles, consequently manifesting as delayed relaxations following voluntary muscle contractions in myotonic subjects. The inheritance pattern of myotonia congenita can be autosomal dominant (Thomsen type) or recessive (Becker type). To date over 200 myotonia-associated ClC-1 mutations have been identified, which are scattered throughout the entire protein sequence. The dominant inheritance pattern of some myotonia mutations may be explained by a dominant-negative effect on ClC-1 channel gating. For many other myotonia mutations, however, no clear relationship can be established between the inheritance pattern and the location of the mutation in the ClC-1 protein. Emerging evidence indicates that the effects of some mutations may entail impaired ClC-1 protein homeostasis (proteostasis). Proteostasis of membrane proteins comprises of biogenesis at the endoplasmic reticulum (ER), trafficking to the surface membrane, and protein turn-over at the plasma membrane. Maintenance of proteostasis requires the coordination of a wide variety of different molecular chaperones and protein quality control factors. A number of regulatory molecules have recently been shown to contribute to post-translational modifications of ClC-1 and play critical roles in the ER quality control, membrane trafficking, and peripheral quality control of this chloride channel. Further illumination of the mechanisms of ClC-1 proteostasis network will enhance our understanding of the molecular pathophysiology of myotonia congenita, and may also bring to light novel therapeutic targets for skeletal muscle dysfunction caused by myotonia and other pathological conditions