Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination.

Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism.

Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins.

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.

Characterization of immunomodulating agents from Staphylococcus aureus for priming immunotherapy in triple-negative breast cancers.

Immunotherapy, specifically immune checkpoint blockade (ICB), has revolutionized the treatment paradigm of triple-negative breast cancers (TNBCs). However, a subset of TNBCs devoid of tumor-infiltrating T cells (TILs) or PD-L1 expression generally has a poor response to immunotherapy. In this study, we aimed to sensitize TNBCs to ICB by harnessing the immunomodulating potential of S. aureus, a breast-resident bacterium. We show that intratumoral injection of spent culture media from S. aureus recruits TILs and suppresses tumor growth in a preclinical TNBC model. We further demonstrate that α-hemolysin (HLA), an S. aureus-produced molecule, increases the levels of CD8+ T cells and PD-L1 expression in tumors, delays tumor growth, and triggers tumor necrosis. Mechanistically, while tumor cells treated with HLA display Gasdermin E (GSDME) cleavage and a cellular phenotype resembling pyroptosis, splenic T cells incubated with HLA lead to selective expansion of CD8+ T cells. Notably, intratumoral HLA injection prior to ICB augments the therapeutic efficacy compared to ICB alone. This study uncovers novel immunomodulatory properties of HLA and suggests that intratumoral administration of HLA could be a potential priming strategy to expand the population of TNBC patients who may respond to ICB.

Human breast microbiome correlates with prognostic features and immunological signatures in breast cancer.

BACKGROUND: Currently, over half of breast cancer cases are unrelated to known risk factors, highlighting the importance of discovering other cancer-promoting factors. Since crosstalk between gut microbes and host immunity contributes to many diseases, we hypothesized that similar interactions could occur between the recently described breast microbiome and local immune responses to influence breast cancer pathogenesis. METHODS: Using 16S rRNA gene sequencing, we characterized the microbiome of human breast tissue in a total of 221 patients with breast cancer, 18 individuals predisposed to breast cancer, and 69 controls. We performed bioinformatic analyses using a DADA2-based pipeline and applied linear models with White's t or Kruskal-Wallis H-tests with Benjamini-Hochberg multiple testing correction to identify taxonomic groups associated with prognostic clinicopathologic features. We then used network analysis based on Spearman coefficients to correlate specific bacterial taxa with immunological data from NanoString gene expression and 65-plex cytokine assays. RESULTS: Multiple bacterial genera exhibited significant differences in relative abundance when stratifying by breast tissue type (tumor, tumor adjacent normal, high-risk, healthy control), cancer stage, grade, histologic subtype, receptor status, lymphovascular invasion, or node-positive status, even after adjusting for confounding variables. Microbiome-immune networks within the breast tended to be bacteria-centric, with sparse structure in tumors and more interconnected structure in benign tissues. Notably, Anaerococcus, Caulobacter, and Streptococcus, which were major bacterial hubs in benign tissue networks, were absent from cancer-associated tissue networks. In addition, Propionibacterium and Staphylococcus, which were depleted in tumors, showed negative associations with oncogenic immune features; Streptococcus and Propionibacterium also correlated positively with T-cell activation-related genes. CONCLUSIONS: This study, the largest to date comparing healthy versus cancer-associated breast microbiomes using fresh-frozen surgical specimens and immune correlates, provides insight into microbial profiles that correspond with prognostic clinicopathologic features in breast cancer. It additionally presents evidence for local microbial-immune interplay in breast cancer that merits further investigation and has preventative, diagnostic, and therapeutic potential.

Using Electromyography to Detect the Weightings of the Local Muscle Factors to the Increase of Perceived Exertion During Stepping Exercise.

Rate of perceived exertion (RPE) is a clinically convenient indicator for monitoring exercise intensity in cardiopulmonary rehabilitation. It might not be sensitive enough for clinicians to determine the patients' physiological status because its association with the cardiovascular system and local muscle factors is unknown. This study used the electromyographic sensor to detect the local muscle fatigue and stabilization of patella, and analyzed the relationship between various local muscle and cardiovascular factors and the increase of RPE during stepping exercise, a common exercise program provided in cardiopulmonary rehabilitation. Ten healthy adults (4 males and 6 females) participated in this study. Each subject used their right bare foot to step up onto a 23-cm-high step at a constant speed until the RPE score reached 20. The RPE, heart rate (HR), and surface EMG of the rectus femoris (RF), vastus medialis, and vastus lateralis were recorded at 1-minute intervals during the stepping exercise. The generalized estimating equations (GEE) analysis indicated that the increase in RPE significantly correlated with the increase in HR, and decrease in median frequency (MF) of the EMG power spectrum of the RF. Experimental results suggest that the increase in RPE during stepping exercise was influenced by the cardiovascular status, localized muscle fatigue in the lower extremities. The weighting of the local muscle factors was more than half of the weighting of the cardiovascular factor.

KLHL20 links the ubiquitin-proteasome system to autophagy termination.

Autophagy is a dynamic and self-limiting process. The amplitude and duration of this process need to be properly controlled to maintain cell homeostasis, and excessive or insufficient autophagy activity could each lead to disease states. Compared to our understanding of the molecular mechanisms of autophagy induction, little is known about how the autophagy process is turned off after its activation. We recently identified KLHL20 as a key regulator of autophagy termination. By functioning as a substrate-binding subunit of CUL3 ubiquitin ligase, KLHL20 targets the activated ULK1 and phagophore-residing PIK3C3/VPS34 and BECN1 for ubiquitination and proteasomal degradation, which in turn triggers a destabilization of their complex components ATG13 and ATG14. These hierarchical degradation events cause the exhaustion of the autophagic pool of ULK1 and PIK3C3/VPS34 complexes, thereby preventing persistent and excessive autophagy activity. Impairment of KLHL20-dependent feedback regulation of autophagy enhances cell death under prolonged starvation and aggravates muscle atrophy in diabetic mice, which highlights the pathophysiological significance of this autophagy termination mechanism in cell survival and tissue homeostasis. Modulation of this autophagy termination pathway may be effective for treating diseases associated with deregulation of autophagy activity.

Cul3-KLHL20 ubiquitin ligase: physiological functions, stress responses, and disease implications.

Cullin-RING ubiquitin ligases are the largest Ubiquitin ligase family in eukaryotes and are multi-protein complexes. In these complexes, the Cullin protein serves as a scaffold to connect two functional modules of the ligases, the catalytic subunit and substrate-binding subunit. KLHL20 is a substrate-binding subunit of Cullin3 (Cul3) ubiquitin ligase. Recent studies have identified a number of substrates of KLHL20-based ubiquitin ligase. Through ubiquitination of these substrates, KLHL20 elicits diverse cellular functions, some of which are associated with human diseases. Furthermore, the functions, subcellular localizations, and expression of KLHL20 are regulated by several physiological and stressed signals, which allow KLHL20 to preferentially act on certain substrates to response to these signals. Here, we provide a summary of the functions and regulations of KLHL20 in several physiological processes and stress responses and its disease implications.

Arecoline N-oxide: its mutagenicity and possible role as ultimate carcinogen in areca oral carcinogenesis.

The areca nut is the most widely consumed psychoactive substance in Taiwan, India, and Southeast Asia. It is considered to be an environmental risk factor for the development of oral submucous fibrosis and cancer. Arecoline, the major alkaloid of areca nut, has been known to cause cytotoxicity and genotoxicity in various systems. However, the active compound accounting for arecoline-induced damage in normal human oral cells is still uncharacterized. The present study was undertaken to identify the active metabolite of arecoline that might induce damage in human oral tissues and cause mutagenicity in Salmonella typhimurium tester strains TA 100 and TA 98. It is interesting to find that the major metabolite of arecoline, arecoline N-oxide, is moderately mutagenic to these Salmonella tester strains. This mutagenicity was potently inhibited by sulfhydryl compounds, namely, glutathione, N-acetylcysteine, and cysteine, whereas methionine is inactive in this inhibition. The mutagenicity of arecoline N-oxide was strongly inhibited by the N-oxide reducing agent titanium trichloride. The possible role of arecoline N-oxide in the induction of oral carcinogenesis by areca nut chewing is discussed.