Role of the HSP70 Co-Chaperone SIL1 in Health and Disease

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1′s function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1′s functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1′s NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.     

Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress,Glucose Regulated Protein 78, and Nanosized Particles

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.     

Unfolding the Unfolded Protein Response: Unique Insights into Brain Ischemia

The endoplasmic reticulum (ER) is responsible for processing of proteins that are destined to be secreted, enclosed in a vesicle, or incorporated in the plasma membrane. Nascent peptides that enter the ER undergo a series of highly regulated processing steps to reach maturation as they transit the ER. Alterations in the intracellular environment that induce ER stress are thought to interrupt these processing steps, and result in unfolding of proteins in the ER. Accumulation of unfolded proteins concurrently activates three transmembrane stress sensors, IRE1, ATF6 and PERK, and is referred to as the Unfolded Protein Response (UPR). Our understanding of the mechanisms of UPR induction has been assembled primarily from experiments inducing ER stress with chemical and genetic manipulations. However, physiological stress often induces activation of ER stress sensors in a distinct manner from the canonical UPR. The unique activation profiles in vivo have prompted us to examine the mechanism of UPR activation in neurons following cerebral ischemia.     

Proteomic Analysis of Marinesco–Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss

Marinesco–Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.     

BiP Heterozigosity Aggravates Pathological Deterioration in Experimental Amyotrophic Lateral Sclerosis

In the present study, we investigated the involvement of the chaperone protein BiP (also known as GRP78 or Hspa5), a master regulator of intracellular proteostasis, in two mouse models of neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD). To this end, we used mice bearing partial genetic deletion of the BiP gene (BiP^+/− mice), which, for the ALS model, were crossed with mutant SOD1 (mSOD1) transgenic mice to generate mSOD1/BiP^+/− double mutant mice. Our data revealed a more intense neurological decline in the double mutants, reflected in a greater deterioration of the neurological score and rotarod performance, with also a reduced animal survival, compared to mSOD1 transgenic mice. Such worsening was associated with higher microglial (labelled with Iba-1 immunostaining) and, to a lesser extent, astroglial (labelled with GFAP immunostaining) immunoreactivities found in the double mutants, but not with a higher loss of spinal motor neurons (labelled with Nissl staining) in the spinal cord. The morphological analysis of Iba-1 and GFAP-positive cells revealed a higher presence of activated cells, characterized by elevated cell body size and shorter processes, in double mutants compared to mSOD1 mice with normal BiP expression. In the case of the PD model, BiP^+/− mice were unilaterally lesioned with the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). In this case, however, we did not detect a greater susceptibility to damage in mutant mice, as the motor defects caused by 6-OHDA in the pole test and the cylinder rearing test, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity (labelled with CD68 and GFAP immunostaining) detected in the substantia nigra were of similar magnitude in BiP^+/− mice compared with wildtype animals. Therefore, our findings support the view that a dysregulation of the protein BiP may contribute to ALS pathogenesis. As BiP has been recently related to cannabinoid type-1 (CB₁) receptor function, our work also opens the door to future studies on a possible link between BiP and the neuroprotective effects of cannabinoids that have been widely reported in this neuropathological context. In support of this possibility, preliminary data indicate that CB₁ receptor levels are significantly reduced in mSOD1 mice having partial deletion of BiP gene.     

一种新型的128路多通道HDLC引擎设计

文章讨论了在SoC通讯处理器芯片中的多通道通讯引擎的结构。该通讯处理器中包括了能处理不同通讯协议的微引擎，多通道微引擎是其中的一个，其实现采用了新的体系结构，4个32路通道独立运作，可支持多种工作模式，全部复用可以处理高达128路通道。每个32路通道内部采用了可重用的结构，分为协议处理为主的位处理器和数据处理为主的字节处理器两大部分。该设计通过了FPGA验证。     

Stellettin B-Induced Oral Cancer Cell Death via Endoplasmic Reticulum Stress–Mitochondrial Apoptotic and Autophagic Signaling Pathway

Oral squamous cell carcinoma (OSCC) affects tens of thousands of people worldwide. Despite advances in cancer treatment, the 5-year survival rate of patients with late-stage OSCC is low at 50–60%. Therefore, the development of anti-OSCC therapy is necessary. We evaluated the effects of marine-derived triterpene stellettin B in human OC2 and SCC4 cells. Stellettin B dose-dependently decreased the viability of both cell lines, with a significant reduction in OC2 cells at ≥0.1 µM at 24 and 48 h, and in SCC4 cells at ≥1 µM at 24 and 48 h. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells were significantly observed at 20 µM of stellettin B at 48 h, with the overexpression of cleaved caspase3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, mitochondrial respiratory functions were ablated by stellettin B. Autophagy-related LC3-II/LC3-I ratio and Beclin-1 proteins were increased, whereas p62 was decreased. At 20 µM at 48 h, the expression levels of the endoplasmic reticulum (ER) stress biomarkers calnexin and BiP/GRP78 were significantly increased and mitogen-activated protein kinase (MAPK) signaling pathways were activated. Further investigation using the autophagy inhibitor 3-methyladenine (3-MA) demonstrated that it alleviated stellettin B-induced cell death and autophagy. Overall, our findings show that stellettin B induces the ER stress, mitochondrial stress, apoptosis, and autophagy, causing cell death of OSCC cells.     

Characterization of deletion mutations in the carboxy-terminal peptide-binding domain of the Kar2 protein in Saccharomyces cerevisiae

Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr₄₉₂–Thr₅₁₂), corresponding to the β8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu₅₁₃–Lys₅₄₂), corresponding to α-helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of α-amylase at non-permissive temperature. © 1998 John Wiley & Sons, Ltd.