Youbao Sha

Autophagy Regulates Phagocytosis by Modulating the Expression of Scavenger Receptors

Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7⁻/⁻) mice. Atg7⁻/⁻ macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7⁻/⁻ mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7⁻/⁻ macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7⁻/⁻ macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.

Functional Variant in the Autophagy-Related 5 Gene Promotor is Associated with Childhood Asthma

Rationale and Objective Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. Methods Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. Measurements and Main Results We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. Conclusion Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.

A critical role for CHIP in the aggresome pathway.

ABSTRACT Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed “physiologic aggresome,” is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIP-mediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTRΔF508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.

COPA mutations impair ER-Golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis

Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4+ T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.

Regulation of NF-κB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated cellular proteins or changes in proteasome expression, suggesting that Rpn13 must have a specialized role in proteasome function. Thus, Rpn13 participation in protein degradation, by recruiting UCH37, is rather selective to specific proteins whose degradation critically depends on UCH37 deubiquitination activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn13 is involved in iNOS degradation and is required for iNOS interaction with the deubiquitination protein UCH37. Furthermore, we discovered that IκB-α, a protein whose proteasomal degradation activates the transcription factor NF-κB, is also a substrate for the Rpn13/UCH37 complex. Thus, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway.

Regulation of IL-4 Receptor Signaling by STUB1 in Lung Inflammation

RATIONALE IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4- and IL-13-mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive. OBJECTIVES To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation. METHODS The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1(-/-) mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease. MEASUREMENTS AND MAIN RESULTS STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation. CONCLUSIONS Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.

STUB1 regulates TFEB‐induced autophagy–lysosome pathway

TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR. The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone‐dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin–proteasome pathway. Phosphorylated TFEB accumulated in STUB1‐deficient cells and in tissues of STUB1‐deficient mice resulting in reduced TFEB activity. Conversely, cellular overexpression of STUB1 resulted in reduced phosphorylated TFEB and increased TFEB activity. STUB1 preferentially interacted with and ubiqutinated phosphorylated TFEB, targeting it to proteasomal degradation. Consistent with reduced TFEB activity, accumulation of phosphorylated TFEB in STUB1‐deficient cells resulted in reduced autophagy and reduced mitochondrial biogenesis. These studies reveal that the ubiquitin–proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.

The E3 ubiquitin ligase STUB1 regulates autophagy and mitochondrial biogenesis by modulating TFEB activity

ABSTRACT TFEB is a master regulator for transcription of genes involved in autophagy, lysosome and mitochondrial biogenesis. Activity of TFEB is inhibited upon its phosphorylation. STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin proteasome pathway. Thus, the ubiquitin-proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.