Systematic analysis reveals a functional role for STAMBPL1 in the epithelial-mesenchymal transition process across multiple carcinomas.

BACKGROUND: Deubiquitinating enzymes (DUBs) are linked to cancer progression and dissemination, yet less is known about their regulation and impact on epithelial-mesenchymal transition (EMT). METHODS: An integrative translational approach combining systematic computational analyses of The Cancer Genome Atlas cancer cohorts with CRISPR genetics, biochemistry and immunohistochemistry methodologies to identify and assess the role of human DUBs in EMT. RESULTS: We identify a previously undiscovered biological function of STAM-binding protein like 1 (STAMBPL1) deubiquitinase in the EMT process in lung and breast carcinomas. We show that STAMBPL1 expression can be regulated by mutant p53 and that its catalytic activity is required to affect the transcription factor SNAI1. Accordingly, genetic depletion and CRISPR-mediated gene knockout of STAMBPL1 leads to marked recovery of epithelial markers, SNAI1 destabilisation and impaired migratory capacity of cancer cells. Reversely, STAMBPL1 expression reprogrammes cells towards a mesenchymal phenotype. A significant STAMBPL1-SNAI1 co-signature was observed across multiple tumour types. Importantly, STAMBPL1 is highly expressed in metastatic tissues compared to matched primary tumour of the same lung cancer patient and its expression predicts poor prognosis. CONCLUSIONS: Our study provides a novel concept of oncogenic regulation of a DUB and presents a new role and predictive value of STAMBPL1 in the EMT process across multiple carcinomas.

Regulation of hyperglycemia in diabetic mice by autolysates from ß-mannanase-treated brewer's yeast.

BACKGROUND: Diabetes mellitus is a serious chronic disease, characterized by hyperglycemia. This study administered either ß-mannanase-treated yeast cell autolysis supernatant (YCS) or yeast cell-wall residues after autolysis (YCR) to investigate their influence on the alleviation of diabetes in a diabetic mouse model. RESULTS: Application of either YCS or YCR led to body weight gain, blood glucose reduction, and an improvement in lipid composition in the diabetic mice. Administration of YCS was more effective in inhibiting oxidative stress than YCR. The expression of PPARα and CPT1α was enhanced, improving lipid biosynthesis, and Trx1 and HIF-1-α genes were downregulated due to the activation of thioredoxin following the interventions, indicating that the processes of lipid metabolism and oxidative stress were heavily involved in the reduction of diabetic characteristics following the interventions. The current study revealed that consumption of YCR also led to a reduction in hyperglycemia, this being associated with its richness in mineral elements, such as chromium and selenium. CONCLUSION: This study may highlight the potential of both YCS and YCR as functional ingredients in dietary formula for improving diabetic syndromes. © 2019 Society of Chemical Industry.

miR-126-5p targets Malate Dehydrogenase 1 in non-small cell lung carcinomas.

Malate Dehydrogenase (MDH) 1 has recently been shown to be highly expressed and display prognostic value in non-small cell lung carcinomas (NSCLCs). However, it is not known how MDH1 expression is regulated and there is no current molecular or chemical strategy that specifically targets MDH1. This may be due to structural and enzymatic similarities with its isoenzyme, malate dehydrogenase 2 (MDH2). However, MDH1 and MDH2 are encoded by distinct genes and this opens up the possibility for modulation at the expression level. Here, we screened in silico for microRNAs (miRs) that selectively targets the 3'UTR region of MDH1. These analyses revealed that mir-126-5p has three binding sites in the 3'UTR region of MDH1. Additionally, we show that expression of miR-126-5p suppresses the enzymatic activity of MDH1, mitochondrial respiration and caused cell death in NSCLC cell lines.

USP10 regulates the stability of the EMT-transcription factor Slug/SNAI2.

Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism governing the switch of cells from an epithelial to a motile mesenchymal-like state. This transdifferentiation is regulated by key transcription factors, including Slug. The stability and function of Slug can be regulated by multiple mechanisms, including ubiquitin-mediated post-translational modifications. Here, by using a genome wide siRNA screen for human deubiquitinating enzymes (DUBs), we identified USP10 as a deubiquitinase for Slug in cancer cells. USP10 interacts with Slug and mediates its degradation by the proteasome. Importantly, USP10 is concomitantly highly expressed with Slug in cancer biopsies. Genetic knockdown of USP10 leads to suppressed Slug levels with a decreased expression of the mesenchymal marker Vimentin. Further, it reduces the migratory capacity of cancer cells. Reversely, overexpression of USP10 elevates the level of both Slug and Vimentin. Our study identifies USP10 as a regulator of the EMT-transcription factor Slug and cell migration.

Gingipains from the Periodontal Pathogen Porphyromonas gingivalis Play a Significant Role in Regulation of Angiopoietin 1 and Angiopoietin 2 in Human Aortic Smooth Muscle Cells.

Angiopoietin 1 (Angpt1) and angiopoietin 2 (Angpt2) are the ligands of tyrosine kinase (Tie) receptors, and they play important roles in vessel formation and the development of inflammatory diseases, such as atherosclerosis. Porphyromonas gingivalis is a Gram-negative periodontal bacterium that is thought to contribute to the progression of cardiovascular disease. The aim of this study was to investigate the role of P. gingivalis infection in the modulation of Angpt1 and Angpt2 in human aortic smooth muscle cells (AoSMCs). We exposed AoSMCs to wild-type (W50 and 381), gingipain mutant (E8 and K1A), and fimbrial mutant (DPG-3 and KRX-178) P. gingivalis strains and to different concentrations of tumor necrosis factor (TNF). The atherosclerosis risk factor TNF was used as a positive control in this study. We found that P. gingivalis (wild type, K1A, DPG3, and KRX178) and TNF upregulated the expression of Angpt2 and its transcription factor ETS1, respectively, in AoSMCs. In contrast, Angpt1 was inhibited by P. gingivalis and TNF. However, the RgpAB mutant E8 had no effect on the expression of Angpt1, Angpt2, or ETS1 in AoSMCs. The results also showed that ETS1 is critical for P. gingivalis induction of Angpt2. Exposure to Angpt2 protein enhanced the migration of AoSMCs but had no effect on proliferation. This study demonstrates that gingipains are crucial to the ability of P. gingivalis to markedly increase the expressed Angpt2/Angpt1 ratio in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. These findings further support the association between periodontitis and cardiovascular disease.

O-GlcNAcylation regulates long-chain fatty acid metabolism by inhibiting ACOX1 ubiquitination-dependent degradation.

BACKGROUND: Cold as a common environmental stress, causes increased heat production, accelerated metabolism and even affects its production performance. How to improve the adaptability of the animal organism to cold has been an urgent problem. As a key hub of lipid metabolism, the liver can regulate lipid metabolism to maintain energy balance, and O-GlcNAcylation is a kind of important PTMs, which participates in a variety of signaling and mechanism regulation, and at the same time, is very sensitive to changes in stress and nutritional levels, and is the body's "stress receptors" and "nutrient receptors". Therefore, the aim of this experiment was to investigate the effect of cold-induced O-GlcNAcylation on hepatic lipid metabolism, and to explore the potential connection between O-GlcNAcylation and hepatic lipid metabolism. METHODS: To investigate the loss of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the precise impacts of additional cold-induced circumstances on liver mass, shape, and metabolic profile, C57 mice were used as an animal model. Using the protein interactions approach, the mechanism of O-GlcNAcylation, as well as the degradation pathway of acyl-Coenzyme A oxidase 1 (ACOX1), were clarified. Additional in vitro analyses of oleic acid (OA) and OGT inhibitor tetraoxan (Alloxan) (Sigma, 2244-11-3) on lipid breakdown in AML-12 cells. RESULTS: In C57BL/6 mice, deletion of O-GlcNAcylation disrupted lipid metabolism, caused hepatic edema and fibrosis, and altered mitochondrial apoptosis. This group of modifications was made worse by cold induction. The accumulation of medium- and long-chain fatty acids is a hallmark of lipolysis, which is accelerated by the deletion of O-GlcNAcylation, whereas lipid synthesis is slowed down. The association between ACOX1 and OGT at the K48 gene precludes ubiquitinated degradation.

The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation.

BACKGROUND: Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function. RESULTS: AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways. CONCLUSIONS: This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.

A Method for Coexpression Analysis.

Gene coexpression network analysis is a commonly used approach in bioinformatics and biomedical research to construct coexpression networks and detect coexpressed genes. This type of analysis has proven valuable for gene function prediction and for disease biomarker discovery.Here, we introduce and guide researchers through a method of differential coexpression analysis focusing on key autophagy and metabolic genes. We utilized the open-source Cancer Cell Line Encyclopedia (CCLE ) project as this is one of the most comprehensive genomic and transcriptomic resources including more than 1000 cell lines of distinct origins. However, the coexpression analysis method described here can also be applied to any open-source dataset where the RNA expression are provided.We here provide detailed comprehensive practical instructions for investigators to successfully identify novel coexpression signatures.

A Method to Identify Potential Prognostic Markers Across Distinct Tumor Types.

The identification of novel biomarkers in cancer patients often requires both survival and gene expression analyses. The Kaplan-Meier survival analysis is one of the most common methods to assess the fraction of subjects living for a certain amount of time.Here, we describe a method for researchers to identify potential prognostic markers across distinct tumor types. We utilize The Cancer Genome Atlas (TCGA) as this is one of the most extensive and successful cancer genomics programs to date that includes expression data and clinical follow-up information for up to 33 distinct tumor types. Nevertheless, the method described here can also be applied to any open-source dataset where the RNA expression and clinical outcome are provided.We provide detailed practical instructions and advices for investigators to be able to successfully identify prognostic markers in cancer patients.

Cold Stress Induced Liver Injury of Mice through Activated NLRP3/Caspase-1/GSDMD Pyroptosis Signaling Pathway.

The body needs to generate heat to ensure basic life activities when exposed to cold temperatures. The liver, as the largest glycogen storage organ in the body and main heat-producing organ at rest, may play a role in chronic cold exposure. Recent studies suggested that pyroptosis plays a crucial role in liver diseases. However, the role of pyroptosis in cold stress-induced liver injury is not clear. Hence, in this study, we attempted to investigate the effects of chronic cold exposure on liver function, apoptosis, oxidative stress and inflammation in mice by establishing a mouse model of chronic cold exposure, and to investigate whether pyroptosis pathways are involved in the process of chronic cold exposure. In vivo, our results show that inflammatory cell infiltration and other pathological changes in liver cells and the activity of liver enzyme evidently increased in the serum and liver of cold-exposed mice, suggesting cold stress may result in liver injury. Remarkably, increased expression of heat shock protein 70 (HSP70) and HSP90 proteins proved the cold stress model is successfully constructed. Then, elevated levels of apoptosis, inflammation, oxidative stress and pyroptosis related proteins and mRNAs, such as cysteinyl aspartate specific proteinase-3 (Caspase-3), inducible nitric oxide synthase (iNOS), nuclear factor erythroid2-related factor 2 (Nrf2) and gasdermins D (GSDMD), confirmed that cold exposure activated apoptosis, oxidative stress and pyroptosis, and released inflammation cytokines. Meanwhile, in vitro, we got similar results as in vivo. Further, adding an NLR family pyrin domain containing 3 (NLRP3) inhibitors found that suppression expression of NLRP3 results in the essential proteins of pyroptosis and antioxidant evidently reduced, and adding GSDMD inhibitor found that suppression expression of GSDMD accompanies with the level of Nrf2 and heme oxygenase-1 (HO-1) obviously reduced. In summary, these findings provide a new understanding of the underlying mechanisms of the cold stress response, which can inform the development of new strategies to combat the effects of hypothermia.

Role of Thioredoxin-1 and its inducers in human health and diseases.

Thioredoxin-1 (Trx-1) is a small redox-active protein normally found in mammalian cells that responds to the changing redox environment by contributing electrons or regulating related proteins. There is growing evidence that Trx-1 has multiple functions, including cytoprotective, anti-apoptotic, antioxidant and anti-inflammatory effects. To date, researchers have found that Trx-1 deficiency leads to severe damage in various disease models, such as atherosclerosis, cerebral ischemia, diabetes and tumors. Conversely, activation of Trx-1 has a protective effect against these diseases. Accordingly, a variety of Trx-1 inducers have been widely used in the clinic with significant therapeutic value. In this paper, we summarize the pathogenesis of Trx-1 involvement in the above-mentioned diseases and describe the protective effects of Trx-1 inducers on them.

Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting.

Autophagic pathways are regulated mechanisms that play important roles in lysosome-mediated cellular degradation. Yet, the contribution of different autophagic pathways in lysosomal targeting, and characterization of the extent and specificity in their degradome remains largely uncharacterized. By undertaking a multiplex quantitative mass spectrometry approach, we have previously analyzed the lysosomal proteome during chaperone-mediated autophagy (CMA)-stimulated conditions in cancer cells. Here, we have extended our multiplex quantitative mass spectrometry and bioinformatics analysis on the proteome from isolated lysosomes to gain a comprehensive view of the temporal enriched lysosomal content upon non-macroautophagy-activated conditions. In parallel, we describe the functional dependency of LAMP2A on, and to what degree the presence of KFERQ-like motifs in proteins influences, their lysosomal targeting. These findings establish a framework for a better understanding of the degradome mediated by autophagic pathways beyond macroautophagy, and present characterization of the impact of LAMP2A in lysosomal targeting in cancer cells.Abbreviations: CMA: chaperone-mediated autophagy; ER: endoplasmic reticulum; EIF4A1: eukaryotic translation initiation factor 4A1; eMI: endosomal microautophagy; FC: fold change; GO: gene ontology; ISR: integrated stress response; LAMP2A: lysosomal associated membrane protein 2A; MA: macroautophagy; MI: microautophagy; MS: mass spectrometry; PCA: principal component analysis; TAX1BP1: Tax1 binding protein 1.

Integrative meta-analysis of gene expression profiles identifies FEN1 and ENDOU as potential diagnostic biomarkers for cervical squamous cell carcinoma.

Cervical carcinoma is a global public health burden. Given that it is usually asymptomatic at potentially curative stages, the development of clinically accurate tests is critical for early detection and individual risk stratification. The present study performed an integrative meta-analysis of the transcriptomes from 10 cervical carcinoma cohorts, with the aim of identifying biomarkers that are associated with malignant transformation of cervical epithelium, and establish their clinical applicability. From among the top ranked differentially expressed genes, flap structure-specific endonuclease 1 (FEN1) and poly (U)-specific endoribonuclease (ENDOU) were selected for further validation, and their clinical applicability was assessed using immunohistochemically stained microarrays comprising 110 tissue cores, using p16 and Ki67 staining as the comparator tests. The results demonstrated that FEN1 expression was significantly upregulated in 65% of tumor specimens (P=0.0001), with no detectable expression in the non-tumor tissues. Furthermore, its expression was significantly associated with Ki67 staining in tumor samples (P<0.0001), but no association was observed with p16 expression or the presence of human papilloma virus types 16/18, patient age, tumor grade or stage. FEN1 staining demonstrated lower sensitivity than p16 (69.3 vs. 96.8%) and Ki67 (69.3 vs. 76.3%); however, the specificity was identical to p16 and higher than that of Ki67 (100 vs. 71.4%).ENDOU staining was consistent with the microarray results, demonstrating 1% positivity in tumors and 40% positivity in non-tumor tissues. Gene set enrichment analysis of cervical tumors overexpressing FEN1 revealed its association with enhanced growth factor signaling, immune response inhibition and extracellular matrix remodeling, whereas tumors with low ENDOU expression exhibited inhibition of epithelial development and differentiation processes. Taken together, the results of the present study demonstrate the feasibility of the integrative meta-analysis approach to identify relevant biomarkers associated with cervical carcinogenesis. Thus, FEN1 and ENDOU may be useful diagnostic biomarkers for squamous cervical carcinoma. However, further studies are required to determine their diagnostic performance in larger patient cohorts and validate the results presented here.

Mogroside V Alleviates Lipopolysaccharide-Induced Neuroinflammation via Inhibition of TLR4-MyD88 and Activation of AKT/AMPK-Nrf2 Signaling Pathway.

As innate immune effector cells in the central nervous system (CNS), microglia not only are essential for the normal development of nervous system but also act on different neurological diseases, including Alzheimer's disease (AD), Huntington's disease (HD), and other neuroinflammatory diseases. Mogroside V (Mog), a natural plant active ingredient and isolated form of Momordica grosvenori, has been shown to possess anti-inflammatory action, but few studies were carried out to investigate the effects of Mog on neuroinflammation. This study aimed to investigate the role of Mog in lipopolysaccharide- (LPS-) induced neuroinflammation and neuronal damage, revealing the underlying mechanisms. Our data indicated that Mog significantly inhibited the LPS-induced production of proinflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-18, IL-6, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and high mobility group box 1 (HMGB1) in BV-2 cells. We found that Mog also suppressed toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), the phosphorylation of mitogen-activated protein kinases (MAPKs), adenosine 5'-monophosphate- (AMP-) activated protein kinase (AMPK), nuclear factor kappa-B (NF-κB), and protein kinase B (AKT). Moreover, Mog also enhanced the expression of γ-glutamyl cysteine synthetase catalytic subunit (GCLC), modifier subunit (GCLM), heme oxygenase-1 (HO-1), and quinine oxidoreductase 1 (NQO1) proteins, mostly depending on the nuclear translation of nuclear factor erythroid-2 related factor 2 (Nrf2). In contrast, pretreatment with inhibitors of AKT can suppress the phosphorylation of AMPK, Nrf2, and its downstream proteins expression. In summary, Mog might play a protective role against LPS-induced neurotoxicity by inhibiting the TLR4-MyD88 and activation of AMPK/AKT-Nrf2 signaling pathway.

The deubiquitinase JOSD2 is a positive regulator of glucose metabolism.

Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado-Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.

The improvement effect of gastrodin on LPS/GalN-induced fulminant hepatitis via inhibiting inflammation and apoptosis and restoring autophagy.

Fulminant hepatitis (FH), characterized by overwhelmed inflammation and massive hepatocyte apoptosis, is a life-threatening and high mortality rate. Gastrodin (GTD), a phenolic glucoside extracted from Gastrodiaelata Blume, exerts anti-apoptosis, and anti-inflammatory activities. In the present study, we aimed to evaluate whether GTD treatment could alleviate lipopolysaccharide and d-galactosamine (LPS/GalN)-induced FH in mice and its potential mechanisms. These data suggested that GTD treatment remarkably protected against LPS/GalN-induced FH by enhancing the survival rate of mice, reducing ALT and AST levels, attenuating histopathological changes, and suppressing interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α secretion. In addition, GTD treatment relieved hepatic apoptosis by the regulation of peroxisome proliferator-activated receptors (PPARs), P53 and caspase-3/9. Furthermore, GTD treatment could significantly inhibit inflammation-related signaling pathways activated by LPS/GalN, including the suppression of nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) and nuclear factor-kappa B (NF-κB) activation. Importantly, GTD treatment effectively restored but not induced LPS/GalN-reduced the expression of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation, as well as the level of pro-autophagy proteins. Taken together, our investigation indicated that GTD played an essential role in liver protection by relieving hepatocyte apoptosis and inflammation reaction, which may be closely involved in the inhibition of NLRP3 inflammasome and NF-κB activation, regulation of apoptosis-related proteins expression, and the recovery of AMPK/ACC/autophagy.

Isoorientin exerts a protective effect against 6-OHDA-induced neurotoxicity by activating the AMPK/AKT/Nrf2 signalling pathway.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is closely associated with oxidative stress. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key transcription factor that regulates oxidative stress. Isoorientin (IOT), as a dietary C-glucosyl flavone derived from rooibos tea, cereals and legumes, is thought to possess multiple pharmacological activities; however, the protective effect of IOT against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells is still poorly understood. The present study focused on investigating whether IOT could ameliorate neurotoxicity and the underlying mechanisms. Our findings indicated that IOT significantly inhibited neurotoxicity reduced apoptotic cell numbers, reactive oxygen species (ROS) overproduction and mitochondrial membrane potential, and modulated the expression of apoptosis-related proteins, including Bcl-2, Bax and caspase-3, which were induced by 6-OHDA. Moreover, IOT also enhanced the expression of the GCLC, GCLM, HO-1, NQO1 and Trx-1 proteins, which mostly depends on the nuclear translation of Nrf2 and reduced expression of the Keap1 protein. IOT significantly increased the phosphorylation of AMPK, ERK, GSK3ß, JNK, PI3K and AKT. In contrast, pretreatment with the inhibitors of AMPK and PI3K/AKT only suppressed the nuclear translocation of Nrf2. In addition, the expression of these proteins was effectively decreased by 6-OHDA, and this effect was reversed by IOT treatment. Importantly, the effect of IOT on improving 6-OHDA induced neurotoxicity was remarkably abrogated by the application of Nrf2 siRNA and, AMPK and PI3K/AKT inhibitors. In summary, IOT might play a protective role against 6-OHDA-induced neurotoxicity by inducing the expression of various antioxidant enzymes via the activation of the AMPK/AKT-Nrf2 signalling pathway.

Human renal fibroblasts are strong immunomobilizers during a urinary tract infection mediated by uropathogenic Escherichia coli.

To prevent the onset of urosepsis and reduce mortality, a better understanding of how uropathogenic Escherichia coli (UPEC) manages to infiltrate the bloodstream through the kidneys is needed. The present study elucidates if human renal interstitial fibroblasts are part of the immune response limiting a UPEC infection, or if UPEC has the ability to modulate the fibroblasts for their own gain. Microarray results showed that upregulated genes were associated with an activated immune response. We also found that chemokines released from renal fibroblasts upon a UPEC infection could be mediated by LPS and triacylated lipoproteins activating the TLR2/1, TLR4, MAPK, NF-κB and PKC signaling pathways. Furthermore, UPEC was also shown to be able to adhere and invade renal fibroblasts, mediated by the P-fimbriae. Furthermore, it was found that renal fibroblasts were more immunoreactive than renal epithelial cells upon a UPEC infection. However, both renal fibroblasts and epithelial cells were equally efficient at inducing neutrophil migration. In conclusion, we have found that human renal fibroblasts can sense UPEC and mobilize a host response with neutrophil migration. This suggests that renal fibroblasts are not only structural cells that produce and regulate the extracellular matrix, but also highly immunoreactive cells.

Targetome analysis of chaperone-mediated autophagy in cancer cells.

Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells. Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin beta; AR7: atypical retinoid 7; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTS: cathepsins; DDX3X: DEAD-box helicase 3 X-linked; EEF2: eukaryotic translation elongation factor 2; EIF4A1: eukaryotic translation initiation factor 4A1; EIF4H: eukaryotic translation initiation factor 4H; GEO: Gene Expression Omnibus; GO: Gene Ontology; GSEA: gene set enrichment analysis; HK2: hexokinase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP: lysosomal-associated membrane protein; LDHA: lactate dehydrogenase A; NES: normalized enrichment score; NFKBIA: NFKB inhibitor alpha; PCA: principle component analysis; PQ: paraquat; S.D.: standard deviation; SUnSET: surface sensing of translation; TMT: tandem mass tags; TOMM40/TOM40: translocase of outer mitochondrial membrane 40.

Characterization of the Role of the Malate Dehydrogenases to Lung Tumor Cell Survival.

Cellular compartmentalization of biochemical processes in eukaryotic cells is critical for many functions including shuttling of reducing equivalents across membranes. Although coordination of metabolic flux between different organelles is vital for cell physiology, its impact on tumor cell survival is not well understood. By using an integrative approach, we have dissected the role of the key metabolic enzymes Malate dehydrogenases (MDH1 and MDH2) to the survival of Non-small Cell Lung Carcinomas. Here, we report that while both the MDH1 (cytosolic) and the MDH2 (mitochondrial) enzymes display elevated levels in patients compared to normal counterparts, only high expression of MDH1 is associated with poor prognosis. We further show that the MDH1 enzymatic activity is significantly higher in NSCLC cells than that of MDH2. Accordingly, genetic depletion of MDH1 leads to significantly higher toxicity than depletion of MDH2. These findings provide molecular insights into the metabolic characteristics of the malate isoenzymes and mark MDH1 as a potential therapeutic target in these tumors.