N. A. Barlev

MIRUMIR: an online tool to test microRNAs as biomarkers to predict survival in cancer using multiple clinical data sets

MIRUMIR: an online tool to test microRNAs as biomarkers to predict survival in cancer using multiple clinical data sets

AMP-activated protein kinase: Structure, function, and role in pathological processes

Recently, AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy balance at cellular and whole-body levels. Due to the involvement in multiple signaling pathways, AMPK efficiently controls ATP-consuming/ATP-generating processes to maintain energy homeostasis under stress conditions. Loss of the kinase activity or attenuation of its expression leads to a variety of metabolic disorders and increases cancer risk. In this review, we discuss recent findings on the structure of AMPK, its activation mechanisms, as well as the consequences of its targets in regulation of metabolism. Particular attention is given to low-molecular-weight compounds that activate or inhibit AMPK; the perspective of therapeutic use of such modulators in treatment of several common diseases is discussed.

PPISURV: a novel bioinformatics tool for uncovering the hidden role of specific genes in cancer survival outcome

Multiple clinical studies have correlated gene expression with survival outcome in cancer on a genome-wide scale. However, in many cases, no obvious correlation between expression of well-known tumour-related genes (that is, p53, p73 and p21) and survival rates of patients has been observed. This can be mainly explained by the complex molecular mechanisms involved in cancer, which mask the clinical relevance of a gene with multiple functions if only gene expression status is considered. As we demonstrate here, in many such cases, the expression of the gene interaction partners (gene ‘interactome’) correlates significantly with cancer survival and is indicative of the role of that gene in cancer. On the basis of this principle, we have implemented a free online datamining tool (http://www.bioprofiling.de/PPISURV). PPISURV automatically correlates expression of an input gene interactome with survival rates on >40 publicly available clinical expression data sets covering various tumours involving about 8000 patients in total. To derive the query gene interactome, PPISURV employs several public databases including protein–protein interactions, regulatory and signalling pathways and protein post-translational modifications.

KMTase Set7/9 is a critical regulator of E2F1 activity upon genotoxic stress.

During the recent years lysine methyltransferase Set7/9 ((Su(var)-3–9, Enhancer-of-Zeste, Trithorax) domain containing protein 7/9) has emerged as an important regulator of different transcription factors. In this study, we report a novel function for Set7/9 as a critical co-activator of E2 promoter-binding factor 1 (E2F1)-dependent transcription in response to DNA damage. By means of various biochemical, cell biology, and bioinformatics approaches, we uncovered that cell-cycle progression through the G1/S checkpoint of tumour cells upon DNA damage is defined by the threshold of expression of both E2F1 and Set7/9. The latter affects the activity of E2F1 by indirectly modulating histone modifications in the promoters of E2F1-dependent genes. Moreover, Set7/9 differentially affects E2F1 transcription targets: it promotes cell proliferation via expression of the CCNE1 gene and represses apoptosis by inhibiting the TP73 gene. Our biochemical screening of the panel of lung tumour cell lines suggests that these two factors are critically important for transcriptional upregulation of the CCNE1 gene product and hence successful progression through cell cycle. These findings identify Set7/9 as a potential biomarker in tumour cells with overexpressed E2F1 activity.

Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism.

Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

KMT Set7/9 affects genotoxic stress response via the Mdm2 axis.

Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome.

Extracellular Proteasomes Are Deficient in 19S Subunits as Revealed by iTRAQ Quantitative Proteomics.

Proteasome‐mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well‐documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ‐based quantitative proteomics (iTRAQ LC/MS‐MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty‐two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP‐PIPs were associated with protein biosynthesis and, unlike CP‐PIPs, were under represented by chaperon/ATP‐binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 232: 842–851, 2017.

Simultaneous EGFP and Tag Labeling of the β7 Subunit for Live Imaging and Affinity Purification of Functional Human Proteasomes

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation, playing important functions in various biological processes. The C-terminus of the β7 (PSMB4) proteasome subunit was tagged with EGFP and with a composite element for affinity purification and TEV cleavage elution (HTBH). When the construct was retrovirally delivered into HeLa cells, virtually all of the β7-EGFP-HTBH fusion protein was found to be incorporated into fully functional proteasomes. This ensured that subcellular localization of the EGFP signal in living HeLa cells could be attributed to β7-EGFP-HTBH within the proteasome complex rather than to free protein. The β7-EGFP-HTBH fusion can, therefore, serve as a valuable tool for in vivo imaging of proteasomes as well as for high-affinity purification of these complexes and associated molecules for subsequent analyses.

TAp73 transcriptionally represses BNIP3 expression

TAp73 is a tumor suppressor transcriptional factor, belonging to p53 family. Alteration of TAp73 in tumors might lead to reduced DNA damage response, cell cycle arrest and apoptosis. Carcinogen-induced TAp73−/− tumors display also increased angiogenesis, associated to hyperactivition of hypoxia inducible factor signaling. Here, we show that TAp73 suppresses BNIP3 expression, directly binding its gene promoter. BNIP3 is a hypoxia responsive protein, involved in a variety of cellular processes, such as autophagy, mitophagy, apoptosis and necrotic-like cell death. Therefore, through different cellular process altered expression of BNIP3 may differently contribute to cancer development and progression. We found a significant upregulation of BNIP3 in human lung cancer datasets, and we identified a direct association between BNIP3 expression and survival rate of lung cancer patients. Our data therefore provide a novel transcriptional target of TAp73, associated to its antagonistic role on HIF signaling in cancer, which might play a role in tumor suppression.

Mass-spectrometric analysis of proteasome subunits exhibiting endoribonuclease activity

Proteasomes function as the main nonlysosomal machinery of intracellular proteolysis and are involved in the regulation of the majority of important cellular processes. Despite the considerable progress that has been made in understanding the functioning of proteasomes, some issues (in particular, the RNase activity of these ribonucleoprotein complexes and its regulation) remain poorly investigated. In this study, we found to several proteins with electrophoretic mobility that corresponds to that of 20S subunits of the core proteasome complex exhibit endoribonuclease activity with respect to the sense and antisense sequences of the c-myc mRNA 3′-UTR. Mass-spectrometric analysis of tryptic hydrolysates of these proteins showed that the samples contained 20S proteasome subunits—α1 (PSMA6), α5 (PSMA5), α6 (PSMA1), and α7 (PSMA3). A number of new phosphorylation sites of α1 (PSMA6) and α7 (PSMA3) subunits were found, and a form of α5 (PSMA5) subunit with a deletion of 20 N-terminal amino-acid residues was identified. The observed differences in the manifestation of endonuclease activity by individual subunits are apparently due to posttranslational modifications of these proteins (in particular, phosphorylation). It was shown that the specificity of RNase activity changes upon proteasome dephosphorylation and under the influence of Ca2+ and Mg2+ cations. It is concluded that posttranslational modifications of proteasome subunits affect the specificity of their RNase activity.