Ubiquitin ligase SYVN1/HRD1 facilitates degradation of the SERPINA1 Z variant/α-1-antitrypsin Z variant via SQSTM1/p62-dependent selective autophagy

SERPINA1/AAT/α-1-antitrypsin (serpin family A member 1) deficiency (SERPINA1/ AAT-D) is an autosomal recessive disorder characterized by the retention of misfolded SERPINA1/AAT in the endoplasmic reticulum (ER) of hepatocytes and a significant reduction of serum SERPINA1/AAT level. The Z variant of SERPINA1/AAT, containing a Glu342Lys (E342K) mutation (SERPINA1^E342K/ATZ), the most common form of SERPINA1/AAT-D, is prone to misfolding and polymerization, which retains it in the ER of hepatocytes and leads to liver injury. Both proteasome and macroautophagy/autophagy pathways are responsible for disposal of SERPINA1^E342K/ATZ after it accumulates in the ER. However, the mechanisms by which SERPINA1^E342K/ATZ is selectively degraded by autophagy remain unknown. Here, we showed that ER membrane-spanning ubiquitin ligase (E3) SYVN1/HRD1 enhances the degradation of SERPINA1^E342K/ATZ through the autophagy-lysosome pathway. We found that SYVN1 promoted SERPINA1^E342K/ATZ, especially Triton X 100-insoluble SERPINA1^E342K/ATZ clearance. However, the effect of SYVN1 in SERPINA1^E342K/ATZ clearance was impaired after autophagy inhibition, as well as in autophagy-related 5 (atg5) knockout cells. On the contrary, autophagy induction enhanced SYVN1-mediated SERPINA1^E342K/ATZ degradation. Further study showed that SYVN1 mediated SERPINA1^E342K/ATZ ubiquitination, which is required for autophagic degradation of SERPINA1^E342K/ATZ by promoting the interaction between SERPINA1^E342K/ATZ and SQSTM1/p62 for formation of the autophagy complex. Interestingly, SYVN1-mediated lysine 48 (K48)-linked polyubiquitin chains that conjugated onto SERPINA1^E342K/ATZ might predominantly bind to the ubiquitin-associated (UBA) domain of SQSTM1 and couple the ubiquitinated SERPINA1^E342K/ATZ to the lysosome for degradation. In addition, autophagy inhibition attenuated the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ cytotoxicity, and the autophagy inducer rapamycin enhanced the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ-induced cell apoptosis. Therefore, this study proved that SYVN1 enhances SERPINA1^E342K/ATZ degradation through SQSTM1-dependent autophagy and attenuates SERPINA1^E342K/ATZ cytotoxicity.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

Systemic deregulation of autophagy upon loss of ALS- and FTD-linked C9orf72

A genetic mutation in the C9orf72 gene causes the most common forms of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein, predicted to be a DENN-family protein, is reduced in ALS and FTD, but its functions remain poorly understood. Using a 3110043O21Rik/C9orf72 knockout mouse model, as well as cellular analysis, we have found that loss of C9orf72 causes alterations in the signaling states of central autophagy regulators. In particular, C9orf72 depletion leads to reduced activity of MTOR, a negative regulator of macroautophagy/autophagy, and concomitantly increased TFEB levels and nuclear translocation. Consistent with these alterations, cells exhibit enlarged lysosomal compartments and enhanced autophagic flux. Loss of the C9orf72 interaction partner SMCR8 results in similar phenotypes. Our findings suggest that C9orf72 functions as a potent negative regulator of autophagy, with a central role in coupling the cellular metabolic state with autophagy regulation. We thus propose C9orf72 as a fundamental component of autophagy signaling with implications in basic cell physiology and pathophysiology, including neurodegeneration.     

Glutamoptosis: A new cell death mechanism inhibited by autophagy during nutritional imbalance

Glutaminolysis plays a critical role in nutrient sufficiency and cell signaling activation in mammalian cells. Unexpectedly, our recent investigations revealed that the unbalanced activation of glutaminolysis during nutritional restriction causes a particular form of apoptotic cell death, that we termed “glutamoptosis.“ We found that the inhibition of autophagy is a key step to allow glutamoptosis-mediated cell death. Thus, autophagy controls glutamoptosis during nutritional imbalance.     

UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in β-cells: Evidence of interplay between the ubiquitin/proteasome system and autophagy

The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in β-cells and increased β-cell apoptosis attributable at least in part to intracellular toxic oligomers of IAPP (islet amyloid polypeptide). β-cells of individuals with T2DM are also characterized by accumulation of polyubiquitinated proteins and deficiency in the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1 [ubiquitin thiolesterase]), accounting for a dysfunctional ubiquitin/proteasome system. In the present study, we used mouse genetics to elucidate in vivo whether a partial deficit in UCHL1 enhances the vulnerability of β-cells to human-IAPP (hIAPP) toxicity, and thus accelerates diabetes onset. We further investigated whether a genetically induced deficit in UCHL1 function in β-cells exacerbates hIAPP-induced alteration of the autophagy pathway in vivo. We report that a deficit in UCHL1 accelerated the onset of diabetes in hIAPP transgenic mice, due to a decrease in β-cell mass caused by increased β-cell apoptosis. We report that UCHL1 dysfunction aggravated the hIAPP-induced defect in the autophagy/lysosomal pathway, illustrated by the marked accumulation of autophagosomes and cytoplasmic inclusions positive for SQSTM1/p62 and polyubiquitinated proteins with lysine 63-specific ubiquitin chains. Collectively, this study shows that defective UCHL1 function may be an early contributor to vulnerability of pancreatic β-cells for protein misfolding and proteotoxicity, hallmark defects in islets of T2DM. Also, given that deficiency in UCHL1 exacerbated the defective autophagy/lysosomal degradation characteristic of hIAPP proteotoxicity, we demonstrate a previously unrecognized role of UCHL1 in the function of the autophagy/lysosomal pathway in β-cells.     

The membrane peroxin PEX3 induces peroxisome-ubiquitination-linked pexophagy

Peroxisomes are degraded by a selective type of autophagy known as pexophagy. Several different types of pexophagy have been reported in mammalian cells. However, the mechanisms underlying how peroxisomes are recognized by autophagy-related machinery remain elusive. PEX3 is a peroxisomal membrane protein (PMP) that functions in the import of PMPs into the peroxisomal membrane and has been shown to interact with pexophagic receptor proteins during pexophagy in yeast. Thus, PEX3 is important not only for peroxisome biogenesis, but also for peroxisome degradation. However, whether PEX3 is involved in the degradation of peroxisomes in mammalian cells is unclear. Here, we report that high levels of PEX3 expression induce pexophagy. In PEX3-loaded cells, peroxisomes are ubiquitinated, clustered, and degraded in lysosomes. Peroxisome targeting of PEX3 is essential for the initial step of this degradation pathway. The degradation of peroxisomes is inhibited by treatment with autophagy inhibitors or siRNA against NBR1, which encodes an autophagic receptor protein. These results indicate that ubiquitin- and NBR1-mediated pexophagy is induced by increased expression of PEX3 in mammalian cells. In addition, another autophagic receptor protein, SQSTM1/p62, is required only for the clustering of peroxisomes. Expression of a PEX3 mutant with substitution of all lysine and cysteine residues by arginine and alanine, respectively, also induces peroxisome ubiquitination and degradation, hence suggesting that ubiquitination of PEX3 is dispensable for pexophagy and an endogenous, unidentified peroxisomal protein is ubiquitinated on the peroxisomal membrane.     

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.     

巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP.利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株.用EGFP标记菌接种小麦'小偃107'种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况.结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦'小偃107'植株在根系发育、株高、分蘖数等方面比对照有较明显的优势.研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用.