Clostridiumnovyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.     

Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.     

RNA-Sequencing Based microRNA Expression Signature of Colorectal Cancer: The Impact of Oncogenic Targets Regulated by miR-490-3p

To elucidate novel aspects of the molecular pathogenesis of colorectal cancer (CRC), we have created a new microRNA (miRNA) expression signature based on RNA-sequencing. Analysis of the signature showed that 84 miRNAs were upregulated, and 70 were downregulated in CRC tissues. Interestingly, our signature indicated that both guide and passenger strands of some miRNAs were significantly dysregulated in CRC tissues. These findings support our earlier data demonstrating the involvement of miRNA passenger strands in cancer pathogenesis. Our study focused on downregulated miR-490-3p and investigated its tumor-suppressive function in CRC cells. We successfully identified a total of 38 putative oncogenic targets regulated by miR-490-3p in CRC cells. Among these targets, the expression of three genes (IRAK1: p = 0.0427, FUT1: p = 0.0468, and GPRIN2: p = 0.0080) significantly predicted 5-year overall survival of CRC patients. Moreover, we analyzed the direct regulation of IRAK1 by miR-490-3p, and its resultant oncogenic function in CRC cells. Thus, we have clarified a part of the molecular pathway of CRC based on the action of tumor-suppressive miR-490-3p. This new miRNA expression signature of CRC will be a useful tool for elucidating new molecular pathogenesis in this disease.     

The Homeobox Only Protein Homeobox (HOPX) and Colorectal Cancer

The HOP (homeobox only protein) homeobox (HOPX) is most closely related to the homeobox protein that contains a homeobox-like domain but lacks certain conserved residues required for DNA binding. Here, we review the current understanding of HOPX in the progression of colorectal cancer (CRC). HOPX was initially reported as a differentiation marker and is expressed in various normal tissues. In the colon, HOPX is expressed uniquely in the quiescent stem cell, +4, and in differentiated mucosal cells of the colon. HOPX expression is markedly suppressed in a subset of cancers, mainly in an epigenetic manner. CRC may include separate entities which are differentially characterized by HOPX expression from a prognostic point of view. HOPX itself can regulate epigenetics, and defective expression of HOPX can result in loss of tumor suppressive function and differentiation phenotype. These findings indicate that HOPX may be both a central regulator of epigenetic dynamics and a critical determinant for differentiation in human cells. HOPX downstream targets were identified in CRC cell lines and hold promise as candidates for therapeutic targets of CRC, such as EphA2 or AP-1. Further analysis will elucidate and confirm the precise role of such proteins in CRC progression.     

Comparative Proteome Research in a Zebrafish Model for Vanishing White Matter Disease

Vanishing white matter (VWM) disease is a genetic leukodystrophy leading to severe neurological disease and early death. VWM is caused by bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (EIF2B). Previous studies have attempted to investigate the molecular mechanism of VWN by constructing models for each subunit of EIF2B that causes VWM disease. The underlying molecular mechanisms of the way in which mutations in EIF2B3 result in VWM are largely unknown. Based on our recent results, we generated an eif2b3 knockout (eif2b3^−/−) zebrafish model and performed quantitative proteomic analysis between the wild-type (WT) and eif2b3^−/− zebrafish, and identified 25 differentially expressed proteins. Four proteins were significantly upregulated, and 21 proteins were significantly downregulated in eif2b3^−/− zebrafish compared to WT. Lon protease and the neutral amino acid transporter SLC1A4 were significantly increased in eif2b3^−/− zebrafish, and crystallin proteins were significantly decreased. The differential expression of proteins was confirmed by the evaluation of mRNA levels in eif2b3^−/− zebrafish, using whole-mount in situ hybridization analysis. This study identified proteins which candidates as key regulators of the progression of VWN disease, using quantitative proteomic analysis in the first EIF2B3 animal model of VWN disease.     

BH3 Mimetic Sensitivity of Colorectal Cancer Cell Lines in Correlation with Molecular Features Identifies Predictors of Response

Colorectal cancer (CRC) is a heterogeneous disease, which in part explains the differential response to chemotherapy observed in the clinic. BH3 mimetics, which target anti-apoptotic BCL-2 family members, have shown potential in the treatment of hematological malignancies and offer promise for the treatment of solid tumors as well. To gain a comprehensive understanding of the response to BH3 mimetics in CRC and the underlying molecular factors predicting sensitivity, we screened a panel of CRC cell lines with four BH3 mimetics targeting distinct anti-apoptotic BCL-2 proteins. Treatment with compounds alone and in combination revealed potent efficacy of combined MCL-1 and BCL-XL inhibition in inducing CRC cell death, irrespective of molecular features. Importantly, expression of the anti-apoptotic protein target of BH3 mimetics on its own did not predict sensitivity. However, the analysis did identify consensus molecular subtype (CMS) specific response patterns, such as higher resistance to single and combined BCL-2 and MCL-1 inhibition in CMS2 cell lines. Furthermore, analysis of mutation status revealed that KRAS mutant cell lines were more resistant to MCL-1 inhibition. Conclusively, we find that CRC cell lines presented with distinct responses to BH3 mimetics that can in part be predicted by their CMS profile and KRAS/BRAF mutations. Overall, almost all CRC lines share sensitivity in the nanomolar range to combined MCL-1 and BCL-XL targeting suggesting that this would be the preferred approach to target these cancers.     

Prognostic Impact and Functional Annotations of KIF11 and KIF14 Expression in Patients with Colorectal Cancer

Genomic instability (GIN) has an important contribution to the pathology of colorectal cancer (CRC). Therefore, we selected mitosis and cytokinesis kinesins, KIF11 and KIF14, as factors of potential clinical and functional value in CRC, as their aberrant expression has been suspected to underlie GIN. We examined the expression and the prognostic and biological significance of KIF11 and KIF14 in CRC via in-house immunohistochemistry on tissue microarrays, public mRNA expression datasets, as well as bioinformatics tools. We found that KIF11 and KIF14 expression, at both the protein and mRNA level, was markedly altered in cancer tissues compared to respective controls, which was reflected in the clinical outcome of CRC patients. Specifically, we provide the first evidence that KIF11 protein and mRNA, KIF14 mRNA, as well as both proteins together, can significantly discriminate between CRC patients with better and worse overall survival independently of other relevant clinical risk factors. The negative prognostic factors for OS were high KIF11 protein, high KIF11 protein + low KIF14 protein, low KIF11 mRNA and low KIF14 mRNA. Functional enrichment analysis revealed that the gene sets related to the cell cycle, DNA replication, DNA repair and recombination, among others, were positively associated with KIF11 or KIF14 expression in CRC tissues. In TCGA cohort, the positive correlations between several measures related to GIN and the expression of KIFs were also demonstrated. In conclusion, our results suggest that CRC patients can be stratified into distinct risk categories by biological and molecular determinants, such as KIF11 and KIF14 expression and, mechanistically, this is likely attributable to their role in maintaining genome integrity.     

Divalent Metal Transporter 1 Knock-Down Modulates IL-1β Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery

Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic β-cells, consequently cell death. Inhibition of β-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced β-cells during IL-1β exposure. Our findings reveal new phosphosites in the IL-1β-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1β exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving β-cell functions upon exposure to IL-1β. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in β-cells after DMT1 silencing.     

Identification of Two Novel Circular RNAs Deriving from BCL2L12 and Investigation of Their Potential Value as a Molecular Signature in Colorectal Cancer

The utility of circular RNAs (circRNAs) as molecular biomarkers has recently emerged. However, only a handful of them have already been studied in colorectal cancer (CRC). The purpose of this study was to identify new circRNAs deriving from BCL2L12, a member of the BCL2 apoptosis-related family, and investigate their potential as biomarkers in CRC. Total RNA extracts from CRC cell lines and tissue samples were reversely transcribed. By combining PCR with divergent primers and nested PCR followed by Sanger sequencing, we were able to discover two BCL2L12 circRNAs. Subsequently, bioinformatical tools were used to predict the interactions of these circRNAs with microRNAs (miRNAs) and RNA-binding proteins (RBPs). Following a PCR-based pre-amplification, real-time qPCR was carried out for the quantification of each circRNA in CRC samples and cell lines. Biostatistical analysis was used to assess their potential prognostic value in CRC. Both novel BCL2L12 circRNAs likely interact with particular miRNAs and RBPs. Interestingly, circ-BCL2L12-2 expression is inversely associated with TNM stage, while circ-BCL2L12-1 overexpression is associated with shorter overall survival in CRC, particularly among TNM stage II patients. Overall, we identified two novel BCL2L12 circRNAs, one of which can further stratify TNM stage II patients into two subgroups with substantially distinct prognosis.     

Changes on the Caco-2 Secretome through Differentiation Analyzed by 2-D Differential In-Gel Electrophoresis (DIGE)

Colorectal cancer is still a major health burden worldwide, and its diagnosis has not improved in recent years due to a lack of appropriate diagnostic serum markers. Aiming to find new diagnostic proteins, we applied the proteomic DIGE technology to analyze changes in the secretome before/after differentiation of the colon adenocarcinoma Caco-2 cell line, an accepted in vitro model to study colorectal tumorigenesis. When the secretomes from undifferentiated (tumor-like) and differentiated cells (resembling healthy enterocytes) were compared, we found 96 spots differentially expressed. After MS/MS analysis, 22 spots corresponding to 15 different proteins were identified. Principal component analysis demonstrated these 22 spots could serve as a discriminatory panel between the tumor-like and normal-like cells. Among the identified proteins, the translationally-controlled tumor protein (TCTP), the transforming growth factor-beta-induced protein ig-h3 (TGFβIp), and the Niemann-Pick disease type C2 protein (NPC2) are interesting candidates for future studies focused on their utility as serum biomarkers of colorectal cancer.     

The Histone Deacetylase Inhibitor BML-210 Influences Gene and Protein Expression in Human Promyelocytic Leukemia NB4 Cells via Epigenetic Reprogramming

Today, cancer is understood as an epigenetic as well as genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. Proteins such as histone deacetylases (HDACs) that cause modifications of histones and other proteins can be targets for novel anticancer agents. Recently, interest in compounds that can inhibit HDACs increased, and now there are many HDACs inhibitors (HDACIs) available with different chemical structures, biological and biochemical properties; hopefully some of them will succeed, probably in combination with other agents, in cancer therapies. In our study we focused on the novel HDACI–BML-210. We found that BML-210 (N-phenyl-Nʹ-(2-Aminophenyl)hexamethylenediamide) inhibits the growth of NB4 cells in dose- and time-dependent manner. In this study we also examined how expression and activity of HDACs are affected after leukemia cell treatment with BML-210. Using a mass spectrometry method we identified proteins that changed expression after treatment with BML-210. We prepared RT-PCR analysis of these genes and the results correlated with proteomic data. Based on these and other findings from our group, we suggest that HDACIs, like BML-210, can be promising anticancer agents in promyelocytic leukemia treatment.     

Urinary Proteomic Signature in Acute Decompensated Heart Failure: Advances into Molecular Pathophysiology

Acute decompensated heart failure (ADHF) is a life-threatening clinical syndrome involving multi-organ function deterioration. ADHF results from multifaceted, dysregulated pathways that remain poorly understood. Better characterization of proteins associated with heart failure decompensation is needed to gain understanding of the disease pathophysiology and support a more accurate disease phenotyping. In this study, we used an untargeted mass spectrometry (MS) proteomic approach to identify the differential urine protein signature in ADHF patients and examine its pathophysiological link to disease evolution. Urine samples were collected at hospital admission and compared with a group of healthy subjects by two-dimensional electrophoresis coupled to MALDI-TOF/TOF mass spectrometry. A differential pattern of 26 proteins (>1.5-fold change, p < 0.005), mostly of hepatic origin, was identified. The top four biological pathways (p < 0.0001; in silico analysis) were associated to the differential ADHF proteome including retinol metabolism and transport, immune response/inflammation, extracellular matrix organization, and platelet degranulation. Transthyretin (TTR) was the protein most widely represented among them. Quantitative analysis by ELISA of TTR and its binding protein, retinol-binding protein 4 (RBP4), validated the proteomic results. ROC analysis evidenced that combining RBP4 and TTR urine levels highly discriminated ADHF patients with renal dysfunction (AUC: 0.826, p < 0.001) and significantly predicted poor disease evolution over 18-month follow-up. In conclusion, the MS proteomic approach enabled identification of a specific urine protein signature in ADHF at hospitalization, highlighting changes in hepatic proteins such as TTR and RBP4.     

Differential expression of liver proteins in streptozotocin-induced diabetic rats in response to hypoglycemic mushroom polysaccharides

We investigated the influence of hypoglycemic fungal extracellular polysaccharides (EPS) on the differential expression of liver proteins in streptozotocin (STZ)-induced diabetic rats. The results of diabetic study revealed that orally administrated EPS exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in EPS-fed rats to 55.1% with enhanced glucose tolerance. In the next step, we analyzed the differential expression patterns of rat liver proteins from each group to discover potent candidates for diabetes-associated proteins. 34 proteins were upregulated and 35 proteins were downregulated upon diabetes induction. Surprisingly, the altered levels of most proteins in the diabetic group were fully or partially restored to those of the non-diabetic control group by EPS treatment. Although the molecular basis of protein modulation after EPS administration in diabetic rats was not verified, the results of the proteomic analysis provide impetus for further studies to identify reliable biomarkers for diabetic therapy.