Proteasomal System Activation And Its Regulation With Autophagic Lysosomal System In HIBM

BackgroundHereditary inclusion body myopathy (HIBM), specially referred as inclusion body myopathy2(IBM2), otherwise known as distal myopathy with rimmed vacuoles (DMRV) was an autosomal recessive characteristic of quadriceps sparing myopathy. It is characterized clinically by early adult onset of muscle weakness in lower distal extremities, slowly progressing in10to20years with relative quadriceps sparing until the late period of disease. HIBM muscle fibers present typical pathology, with rimmed vacuoles formation, grouped atrophic fibers, no dominant inflammatory cells infiltration or necrosis, and tubulo-filamentous inclusions measuring15to20nm in diameter. This disease was caused by mutations in UDP-N-acetylglucosamine2-epimerase/N-acetylmannosamine kinase (GNE) gene located in9p13.2. The UDP-N-acetylglucosamine2-epimerase/N-acetylmannosamine kinase (GNE/MNK) is a generally expressed soluble protein located in cytoplasm, Golgi complex and nuclear, which plays a critical role in sialic acid (Neu5Ac) biosynthesis. Neu5Ac was the most redundant sialic acid in mammalian. It is the classical terminal monosaccharide on glycoconjugates that plays an important role in cellular signaling transduction. The activated form of sialic acid, cytidine monophosphate (CMP)-sialic acid, is utilized as substrate for sialyltransferases by the Golgi complex in the sialylation of glycoconjugates.Although the GNE gene responsible for hereditary inclusion body myopathy (HIBM) has been identified, the exact cellular mechanisms behind the development of muscle fiber degeneration in HIBM have remained elusive. The HIBM associated GNE mutations cause decrease of both enzyme activities leading to decrease of sialic acid production. Although studies on hyposialylation in muscle cells of HIBM have had varied results and whether hyposialylation cause muscle weakness was still disputing, hyposialylation was still the critical factor in the pathogenesis of HIBM. Some glycoconjugates, for example N-linked, might be more readily sialylated than others, for example O-linked or PSA linked. Thus, when a shortage of sialic acid occurs, specific proteins may be inadequately glycosylated, such as PSA-NCAM or alpha-dystroglycan, contributing to the pathology of HIBM.Sialic acids are the most important terminal sugar on glycoconjugates, and play a critical role in proper folding of protein. While hyposialylation can disturb folding of proteins to some extent and result in misfolded proteins. Because amyloid itself is a glycoprotein, when sialic acid was insufficient, amyloid misfolding occurred and misfolded amyloid protein accumulated in endoplasmic reticulum. Intracellular beta amyloid deposition in myofibers has been documented only in few muscle diseases, namely HIBM and sporadic inclusion body myositis (sIBM). Pathologically, the features of HIBM are very similar to those of sIBM, except for the presence of inflammation in sIBM. The role of amyloid deposition in the pathogenesis of muscle diseases has been highlighted in a sIBM mouse model, in which a correlation of intracellular amyloid levels and motor weakness was seen. Amyloid deposition was confirmed as an early pathological finding before rimmed vacuoles and inclusion body formation in a HIBM mouse model. Amyloid deposition in muscle fibers might initiate the downstream pathological events in HIBM, but the process by which how beta-amyloid deposits initiate various degradative pathways and cause the typical pathologic manifestations such as rimmed vacuoles in the pathogenesis have not been clarified.Unfolded polypeptide chains pose an intrinsic threat to the cell's well-being. They are prone to aggregation, which adversely affects redox status, free Ca2+levels, and membrane permeability, leading to cell damage and ultimately cell death. In addition to the chaperones of the heat shock protein family, which shield unfolded proteins from the rest of the cytosol and assist in their refolding, components of the ubiquitin(Ub)/proteasome system have been recognized to play an important role in endoplasmic reticulum associated degradation (ERAD) to protect the cell from the negative effects of protein misfolding and aggregation. Removal of misfolded proteins depends on their recognition in the endoplasmic reticulum (ER) followed by their retrograde transport into the cytosol for degradation. The AAA-ATPase Cdc48/p97 (valosin containing protein, VCP) facilitates the translocation of misfolded ER-proteins into the cytosol. VCP can dock onto the ER-membrane via direct interaction with ER-membrane proteins and/or indirectly via its substrate-recruiting cofactors, which interact with the ubiquitylated substrates at the membrane. This tight interaction in conjunction with the conformational changes induced upon ATP hydrolysis within VCP is thought to provide the driving force for the translocation reaction.The26S proteasome is a700kd proteolytic organelle found throughout the cytoplasm and nuclear of eukaryocyte that consists of a20S catalytic core and two19S regulation complexes.26S proteasome plays an important role in degradation of normal and abnormal proteins via ubiquitin mediated and ATP dependent manner. The19S complexes bind polyubiquited proteins and control access to the proteolytic inner core of20S subunit. The20S proteolytic core consist of a and β subunits. The catalytic activities of β subunits included trypsin-, chymotrypsin-and peptidyl-glutamyl peptide-hydrolyzing-like.26S proteasome system involved of kinds of proteins degradation, including ubiquitinated misfolded and unfolded proteins.However, oligomerization and aggregation of unfolded polypeptides preclude the proteasome from degrading. Autophagy, the mechanism by which cells destroy bulky targets including complete organelles, seems to be of significance in clearance of protein aggregates. The ubiquitin proteasome system and autophagic-lysosomal system are two cornerstones of cellular catabolism that are involved in most aspects of normal physiology and development, and are also implicated in a broad array of pathological states, including cancer, neurodegeneration and aging. Ubiquitin proteasome system and/or autophagy are involved in the degradation of virtually every type of surplus, dysfunctional or damaged cellular component, ranging from soluble proteins to whole organelles. Thus, autophagy and the ubiquitin proteasome system are critical in the maintenance of cellular homeostasis, suggesting that their activities need to be carefully orchestrated. Yet, the two pathways differ so significantly with respect to their mechanistic details (autophagy is a vesicular trafficking pathway, while the enzymatic reactions of the ubiquitin proteasome system occur directly in the cytosol), substrates (the activity of ubiquitin proteasome system is restricted to soluble proteins, while autophagy is practically omnivorous), specificity, elements of control. For a long time these processes were viewed as independent of each other. Here, more and more evidences generated during recent years indicated an unexpected interplay between these two cellular waste conveyors. Autophagy is often thought of as a non-specific process that degrades cytoplasmic proteins and organelles in bulk. Furthermore, recent studies have revealed interaction between the ubiquitin proteasome system and autophagy, suggesting a coordinated and complementary relationship between these degradation systems that becomes critical in times of cellular stress. Specially, these degradation systems share certain substrates and regulatory molecules, and show coordinated and compensatory function. Although the mechanism whereby autophagy and UPS function are coordinated is little understood, several regulators have emerged as important players in mediating this crosstalk, including histone deacetylase6(HDAC6), p62/sequestosome1(p62) and neighbor of BRCA1gene (NBR1). HDAC6interacts with polyubiquitinated proteins through a highly conserved Zn-finger ubiquitin-binding domain, and also interacts with dynein motors, suggesting that the molecule may provide a physical link between ubiquitinated cargo and transport machinery. p62is another cytosolic protein whose structure suggests a function as an adaptor molecule linking ubiquitinated proteins to autophagic machinery. The C-terminal portion of p62harbors both an ubiquitin-associated (UBA) domain which interacts non-covalently with ubiquitinated proteins as well as an LC3-interacting region (LIR). Cellular stresses such as polyglutamine expression, proteasome impairment, oxidative stress, and increased misfolded protein burden activate transcription and translation of p62, suggesting that it functions broadly in stress situations. NBR1is an ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. The pioneering work identified NBR1as another carrier of ubiquitinated proteins for their degradation and an autophagic cargo receptor that is degraded by autophagy. NBR1is considered to cooperate with p62in selective autophagy of ubiquitinated proteins; it has been shown to physically interact with p62, but each one can also work independently.The most typical pathologic findings rimmed vacuoles observed in muscle biopsy of HIBM are actually cluster of autophagic vacuoles and myeloid bodies. The autophagic mature of these vacuoles was supported by the presence of acid-phosphatase-rich primary lysosomes, dathrin positive granules, and the presence of cathepsins B and L in fibers with RVs. Autophagy system activation in HIBM has also been verified by numbers of studies and investigations. Nevertheless, the mechanism of autophagic vacuoles observed in HIBM, known as rimmed vacuoles formation was still unknown. And the correlation between the two degradation pathways (ubiquitin proteasome system and autophagic lysosomal system) in HIBM was obscure.We chose Chinese HIBM patients confirmed by GNE analysis from patients clinically and pathologically diagnosed as HIBM. Our studies will investigate amyloid deposition and muscle fibers'response to that in Chinese HIBM patients confirmed by GNE mutations to elucidate the contribution of transportation of unfolded proteins from endoplasmic reticulum to cytoplasm and the activation of ubiquitin proteasome system in the pathogenesis of HIBM. In an attempt to broaden our understanding of the roles and correlation of ubiquitin proteasome system and autophagic lysosomal system involved in abnormal protein degradation in HIBM muscle fibers, we have also studied the linkers between ubiquitin proteasome system and autophagic lysosomal system including HDAC6, p62and NBR1.Subjects and MethodsSubjects:Patients diagnosed as HIMB by clinical manifestation, muscle pathology and electron microscope in neuromuscular disorder laboratory of Shandong University were selected to test GNE mutations by DNA sequencing analysis. Ten patients were finally confirmed to have GNE mutations by DNA sequencing analysis (formal seven patients confirmed before, recent three patients confirmed for the first time). Five muscle specimens from patients clinically diagnosed as mitochondrial encephalomyopathy for convulsion and confirmed as non-myopathy by muscle pathology and mtDNA analysis were used for normal controls.Methods:1. Genomic DNA was extracted from muscle biopsies by DNA extraction kit and polymerase chain reaction (PCR) amplification was performed to amplify all the11coding exons and their flanking intron sequences of GNE gene. DNA fragments were analyzed by the agarose gel electrophoresis. All the aimed fragments were purified and analyzed by direct gene sequencing, and then comparison was made to detect gene mutations. Once a new mutation was found, more than100unrelated individuals with no history of neuromuscular diseases were tested by restriction enzyme analysis using mismatch primers to evaluate it.2. Immunofluorescence (IF) was performed on4μm thick fresh frozen muscle biopsy samples. To block nonspecific binding of antibody to Fc receptors, sections were pre-incubated with confining liquid containing1%BSA. Sections were then incubated using the well-characterized primary antibodies and rhodamine labelled second anti-mouse antibody. Following washing in0.01PBS, the sections were sealed with0.1M soda-sodium bicarbonate glycerol buffer and observed by fluorescence microscope.3. Western blot:Briefly, about50pieces of10-μm-thick sections of frozen muscle were collected at-25℃and rapidly homogenized on ice with cell lysis buffer for western and IP kits. Twenty to sixty micrograms of protein was loaded into a10%-12%polyacrylamide gel, separated by electrophoresis, and then transferred to a nitrocellulose membrane. Nitrocellulose membranes were blocked in5%(w/v) blocking reagent in phosphate-buffered saline, and were incubated overnight at4℃with primary antibodies. After being washed by PBST, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase at37℃for one hour. The blots were developed using an enhanced chemiluminescence system. The expression of protein was analyzed by Quantity One soft. Protein loading was evaluated by a-skeletal actin or GAPDH band visualized with a monoclonal antibody.4. Proteasome protease activities test: HIBM and control muscle biopsies were homogenized in special cell lysis buffer and centrifuged, the supernatant collected, and protein concentration determined using the BCA method. Subsequently,200μg of biopsied muscle protein were incubated in100μmol/L fluorogenic substrates for the three different protease activities (substrate II for PGPH; substrate III for CTL; substrate IV for TL) for1hour at37℃. Fluorescence emission was excited at360nm and recorded at430nm.5. Real-time quantitative RT-PCR:Total RNA was isolated from30pieces of5-μm-thick sections of frozen muscle biopsy tissues using TRIzol reagent. The total RNA was reverse-transcribed with a RT-PCR kit. The real-time PCR was performed with the use of SYBR Green I kit following the manufacturer's instructions. The relative expression levels of each targeted gene were normalized by subtracting the corresponding beta-actin threshold cycle (CT) values by using the DDCT comparative method.Results1. One p.I587T homozygous mutation and two compound heterozygous mutations of which one was compound of p.Y166H and p.D515fsX2and the other p.D176V and p.V305F were found in three recent clinically diagnosed HIBM patients by GNE sequencing analysis. p.Y166H, p.V305F and p.D515fsX2were new mutations. The two new point mutations were not found in100healthy controls by restricted enzyme analysis.2. Amyloid protein precursor was accumulated in fibers of HIBM. The accumulation of amyloid precursor protein present in two types:patchy accumulation in atrophic fibers and round-like accumulation in normal-size muscle fibers. There was only nonspecific staining in interstitial of normal muscle controls.3. Highly expressed VCP facilitated abnormal folded proteins transporting from cytoplasm to ubiquitin proteasome system. Densitometry analysis of immunoblotting revealed that VCP was increased108.3%(P＜0.05) in HIBM patients than normal controls. Real-time PCR analysis showed VCP was increased215.6%(P<0.05) in HIBM patients than normal controls.4. Proteasome subunits expression was increased and ubiquitin proteasome system was activated. The densitometry of20S proteasome subunits a2, a4and β5evaluated after normalization to the corresponding actin revealed, as compared to control muscle biopsy, that a2was increased162.3%, a4was increased66.3%and P5was increased51.7%(P<0.05). Real-time PCR analysis showed the expression of a2, a4and P5in mRNA level was increased by156.4%,56.7%and96.3%relatively(P＜0.05).5. The activities of three main proteasome protease activities were increased in HIBM. In HIBM biopsies, CTL, TL, and PGPH protease activities were increased48.2%,55.3%, and27.5%(P<0.05), respectively, as compared to the control biopsies. The increase of three main proteasome protease activities further confirmed ubiquitin proteasome system activation in HIBM.6. Expression HDAC6by immunoblotting was increased in HIBM muscle biopsies as compared to control biopsies. In four HIBM muscle biopsies, densitometry analysis of immunoblotting revealed that HDAC6was increased98.6%(P<0.05). Real-time PCR analysis showed that HDAC6was increased135.4%(P<0.05) in HIBM in mRNA level.7. Expression of p62by immunoblotting was increased in HIBM muscle biopsies as compared to control biopsies. In four HIBM muscle biopsies, densitometry analysis of immunoblotting revealed that p62was increased124.8%(P<0.05). Real-time PCR analysis showed that P62was increased64.5%(P<0.05) in HIBM in mRNA level.8. Expression of NBR1by immunoblotting was increased in HIBM muscle biopsies as compared to control biopsies. In four HIBM muscle biopsies, densitometry analysis of immunoblotting revealed that NBR1was increased149.2%(P<0.05). Real-time PCR analysis showed that NBR1was increased95.1%(P<0.05) in HIBM in mRNA level.Conclusions1. High similarity was observed in clinical manifestations of Chinese HIBM patients, while types of GNE mutations were more complicated and highly coincident with their clinical diagnosis. The possible common mutation in Chinese HIBM patients is p.L508S.2. Patchy beta-amyloid precursor protein accumulated in small degenerated atrophic muscle fibers of HIBM patients indicated excessive abnormal folded proteins accumulation in cytoplasm might cause muscle fibers degenerating. Round-like beta-amyloid precursor protein accumulated in normal-size fibers indicated amyloid protein accumulation was the upstream pathologic event in HIBM.3. Highly expressed VCP in muscle fiber of HIBM suggested increased delivery of misfolded or unfolded proteins from endoplasmic reticulum to cytoplasm. The increase expression of20S proteasome subunits in muscle fiber of HIBM according with increase of three main proteasome protease activities indicated ubiquitin proteasome system was activated to participate in the endoplasmic reticulum associated degradation.4. The increase expression of linkers between ubiquitin proteasome system and autophagic lysosomal system indicated an enhanced transportation of ubiquitinated proteins from ubiquitin proteasome system to autophagic lysosomal system. Ubiquitin proteasome system and autophagic lysosomal system coordinated to eliminate the abnormal folded proteins in HIBM. Excessive ubiquitinated unfolded proteins were exported by linkers between UPS and autophagy to autophagy lysosomal system which was activated as an alternative viable pathway for unfolded proteins degradation. 5. Ubiquitin proteasome system activation in endoplasmic reticulum associated degradation and the coordination between ubiquitin proteasome system and autophagic lysosomal system contributes to the pathogenesis of HIBM which might provid new therapeutic point for treating HIBM.