V. L. Karpov

Identification of Proteins with Altered Expression in Colorectal Cancer by Means of 2D-Proteomics

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. Coloreetal cancer is nearly asymptomatic at the early stages, which calls for development of fast and sensitive methods for molecular diagnostics. Proteomes of 11 paired specimens of primary colorectal tumors and adjacent histologically normal tissues were studied using comparative 2D PAGE. Altogether, 16 proteins with altered expression levels were detected, including 13 proteins with increased levels and three proteins with decreased levels in tumor tissues. These proteins were identified using MALDI-TOF mass spectrometry. The proteins GPD1, RRBP1 (increased levels), HNRNPH1, and SERPINB6 (decreased levels) have been associated with colorectal cancer for the first time.

Rpn4p is a positive and negative transcriptional regulator of the ubiquitin-proteasome system

In eukaryotes, the ubiquitin-proteasome proteolytic system is involved in metabolizing most of cell proteins and in major regulation pathways. Although its structure and functioning are fairly well studied, little is known about the regulation of gene expression in this system. The regulation of proteasome gene expression has been described only for Saccharomyces cerevisiae and includes the proteasome-associated transcription factor Rpn4p and its binding site, the proteasome-associated control element (PACE). There are two questions concerning the role of Rpn4p as a transcription factor: whether Rpn4p regulates the PACE-containing genes of the protein ubiquitination system and what contribution Rpn4p makes to the stress-induced changes in proteasome mRNA levels. Semiquantitative RT-PCR showed that deletion of S. cerevisiae RPN4 decreased the RAD6, RAD23, and CDC48 mRNA levels, while the UBI4 mRNA level increased. Stress factors, such as heat shock or the alkylating agent methyl methanesulfonate, induced Rpn4p-dependent transcriptional upregulation of RPT4 and RPN5. At the same time, methyl methanesulfonate downregulated the CDC48 expression in the wild-type strain. Apparently, Rpn4p acts both as an activator and a repressor of transcription of the ubiquitinproteasome genes under normal and stress conditions.

Structure and function of enteric α-defensins in norm and pathology

The review summarizes the current data on the structure of enteric α-defensins, their functions in innate and adaptive immunity systems, and their role in intestinal illnesses.

Escherichia coli Dam-methylase as a molecular tool for mapping binding sites of the yeast transcription factor Rpn4

Rpn4p is a transcription factor responsible for coordinated regulation of proteasomal genes in Saccharomyces cerevisiae. There are data suggesting an involvement of this factor in regulation of many other genes that comprise more than one tenth part of the yeast genome. Traditional methods are inapplicable for mapping of Rpn4p binding sites because of their extremely low level. We have developed a model system using Dam-methylase of E. coli which allows us to detect interaction of Rpn4p with its target genes. In this system, we have shown that Rpn4p is recruited to proteasomal genes only through interactions with DNA.

Proteomic expression analysis of human colorectal cancer: Identification of soluble overexpressed proteins

Colorectal cancer is one of the most common malignancies in developed countries. Scarce clinical signs at the early stages of the disease and the lack of fast and sensitive diagnostic techniques based on the detection of tumor specific protein markers contribute greatly to the high mortality rate. The search for such markers is significantly complicated by the high levels of major structural and cytoskeletal proteins in normal and tumor tissues. Extraction with 0.2 M NaCl in the presence of the nonionic detergent NP-40 was performed to enrich the soluble protein fraction. This modification resulted in a considerably increased sensitivity of detection of minor proteins that may enter the circulation during carcinogenesis. The soluble protein profiles of the paired colon adenocarcinoma and normal tissue specimens were compared using 2D gel electrophoresis, which enabled the detection of 10 proteins whose levels in tumors were elevated at least 10-fold as compared to normal tissue. The proteins were identified by MALDI-TOF mass spectrometry, and two new protein markers of colon cancer, TAF9 and CISH, were discovered. Low levels of CISH synthesis in most normal human tissues and tumors other than colorectal cancer make it a prospective candidate diagnostic marker for this type of cancer.

Mapping of the Rpn4p regions responsible for transcriptional activation of proteasome genes

Rpn4p is an extremely short-lived proteasome-associated protein that acts both as a positive and negative transcriptional regulator of the ubiquitin-proteasome system and as its substrate. The mechanisms of proteasomal degradation of Rpn4p have been studied in great detail; however, the mechanisms of its own action remain unclear and, first of all, its functional domains are unknown. To map the functionally important regions, a set of Rpn4p deletion derivates was constructed. The mutant proteins were expressed in Saccharomyces cerevisiae strain rpn4-Δ, their contents were determined by Western blotting, and their activity was assessed by measuring the mRNA levels of proteasome genes. Deletions of the C-terminal region, containing DNA-binding zinc finger domains, and the N-terminal region, having no homology to the transactivation domains of any known transcription factor, completely inactivated Rpn4p. Only one of the two acidic regions, putative transactivation domains, proved to participate in transcriptional activation. Deletions of the N-terminal region, NAD, or zinc finger domains rendered Rpn4p metabolically stable. These data provide new insights into the mechanisms of the Rpn4p functioning and degradation.

Chromatin structure and transcription regulation in Saccharomyces cerevisiae

The nucleosome organization of eukaryotic chromatin determines the DNA accessibility for regulatory factors during transcription. Nucleosome positioning at promoters is an important factor that affects gene expression. Recent studies showed that yeast promoters can be divided into two groups differing in chromatin organization. It became clear that genes with similar chromatin structures in the promoter regions are regulated by the same mechanisms. The review discusses the interplay between the chromatin structure and transcription, dynamic changes in chromatin during transcription, and the roles of various factors (histone chaperones, remodeling complexes, and the histone variant H2A.Z) in these processes with the example of Saccharomyces cerevisiae.

Identification of proteins overexpressed in papillary thyroid tumors

A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.

Availability and canonical positioning of key amino acids of ornithine-decarboxylase degron is insufficient for alpha-fetoprotein degradation

Ornithine decarboxylase (ODC) degrades in proteasome in a ubiquitin-independent manner with the half life of approximately 2 h. Thirty seven C-terminal amino acids of this enzyme constitute a fragment known as the degradation signal (degron), which is responsible for the effectiveness of protein degradation. Among these amino acids, the key positions have recently been mapped (Cys441 and Ala442). Mutations of the key amino acids led to ODC general stabilization, whereas substitution of other amino acids had no significant influence on the ODC degron activity. In addition, deletions or insertions into the region located between the key amino acids and ODC C-end diminished significantly the rate of protein degradation; hence, the distance (remoteness) of these amino acids from ODC C-end is, probably, of crucial importance. Taking into account these data, we have introduced the key amino acids that determine ODC-degron activity into alpha-fetoprotein with the truncated export signal (ΔAFP) so that their positioning was 20 amino-acid away from the C-end (ΔAFPCAG and ΔAFPLCAG). Secretion of ΔAFP and the modified proteins from cells was impossible because of a removal of the N-terminal export signal. Computer analysis of ΔAFP and the derivative ΔAFPCAG and ΔAFPLCAG revealed no significant changes in protein hydrophobicity or in the secondary structure of C-terminal region. The in vitro experiments on HEK293T cells using MG132 proteasome inhibitor and translation inhibitor cycloheximide have demonstrated similar stability of ΔAFP and the derivative ΔAFPCAG and ΔAFPLCAG in cells. Thus, introduction of the key amino acids of ODC degron at the key positions relative to the C-end of ΔAFP did not change the parameters of protein degradation. Perhaps, some other still unknown amino acids are important for ODC-degron functioning. It may well be that ΔAFP conformation prevents interaction of the protein C-end with proteasome.

Search for protein markers for serum diagnostics of tumors by analysis of microRNA expression profiles

New algorithm of bioinformatic search for potential serum tumor markers has been worked out, including: (1) identification of microRNAs with the level of synthesis most evidently and often decreasing in tumors; (2) search for target mRNAs regulated by microRNAs; (3) selection of targets encoding secretory proteins; (4) comparative analysis of transcription level of the targets in normal and tumor tissues. Practical use of the algorithm has allowed us to detect seven potential serum markers of colon tumors: ADAMTS14, ANGPT2, CCL7, DEFA5, MMP11, MMP14, and PLAU. It has been experimentally indicated that the level of synthesis of two of seven proteins (MMP14 and DEFA5) is significantly increased in colon tumors compared to normal tissue.