Joseph Zaretsky

Generation of ligand-receptor alliances by "SEA" module-mediated cleavage of membrane-associated mucin proteins

A mechanism is described whereby one and the same gene can encode both a receptor protein as well as its specific ligand. Generation of this receptor–ligand partnership is effected by proteolytic cleavage within a specific module located in a membrane resident protein. It is postulated here that the “SEA” module, found in a number of heavily O‐linked glycosylated membrane‐associated proteins, serves as a site for proteolytic cleavage. The subunits generated by proteolytic cleavage of the SEA module reassociate, and can subsequently elicit a signaling cascade. We hypothesize that all membrane resident proteins containing such a “SEA” module will undergo cleavage, thereby generating a receptor–ligand alliance. This requires that the protein subunits resulting from the proteolytic cleavage reassociate with each other in a highly specific fashion. The same SEA module that serves as the site for proteolytic cleavage, probably also contains the binding sites for reassociation of the resultant two subunits. More than one type of module can function as a site for proteolytic cleavage; this can occur not only in one‐pass membrane proteins but also in 7‐transmembrane proteins and other membrane‐associated proteins. The proposal presented here is likely to have significant practical consequences. It could well lead to the rational design and identification of molecules that, by binding to one of the cleaved partners, will act either as agonists or antagonists, alter signal transduction and, hence, cellular behavior.

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen: A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor‐associated antigen, H23, was compared to expression of genes coding for pS2, c‐erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non‐breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c‐erbB2, ER and pS2, respectively. Non‐malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.

MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERα) in regulation of the MUC1 gene expression

BackgroundThe MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter.ResultsUsing TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERα, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed.ConclusionAnalysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERα in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

Analysis of the promoter of the MUC1 gene overexpressed in breast cancer.

The MUC1 gene encodes a mucin glycoprotein and is overexpressed in breast cancer. Knowledge of the mechanisms leading to MUC1 overexpression may help in the development of molecular approaches for breast cancer therapy. In order to study the regulation of the MUC1 gene transcription, we analyzed functional activities of various deletion mutants of the MUC1 promoter. We established that transcriptional cis‐elements present in the SacI/XmnI fragment of the promoter are competent and sufficient for expression of, at least, tandem repeats containing isoform(s) of the MUC1 protein. CAT transfection analysis showed that both the 3′ and 5′ regions of the SacI/XmnI fragment possess transcription activities. Promoter activities associated with the SacI/XmnI fragment were confirmed by a RNase protection assay, which demonstrated multiple transcription start sites (TSSs) in the MUC1 gene transcribed in epithelial T47D cells. We show that treatment of the T47D cells with TGFβ1 leads to activation of additional TSSs in the MUC1 gene. The roles of the structural and functional properties of the MUC1 promoter in MUC1 gene transcription are discussed.

A Novel Protein Derived from the MUC1 Gene by Alternative Splicing and Frameshifting

Genes that have been designated the name “MUC” code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.

Expression of a gene coding for breast tumor-associated antigen in thyroid papillary carcinoma

The expression of the H23 gene, previously shown to be overexpressed in breast cancer tissue, was examined in various thyroid pathologies. Thyroid papillary carcinomas demonstrate significant H23 mRNA levels, whereas benign thyroid pathologies have very low levels of expression. H23 gene expression in thyroid cancer inversely correlated with that of thyroperoxidase and thyroglobulin genes. Immunoblot assays of thyroid cancer tissues revealed overexpression of H23 gene at the protein level as well The data presented indicate that dedifferentiation of thyroid tissue to the malignant state is associated with increased H23 gene expression and suppression of some thyroidal differentiation marker genes.

Protein Multifunctionality: Principles and Mechanisms

In the review, the nature of protein multifunctionality is analyzed. In the first part of the review the principles of structural/functional organization of protein are discussed. In the second part, the main mechanisms involved in development of multiple functions on a single gene product(s) are analyzed. The last part represents a number of examples showing that multifunctionality is a basic feature of biologically active proteins.

Molecular Analysis of H23 Epithelial Tumor Antigen - Differentially Spliced Full Length cDNAs and Gene

The isolation and characterization of the complementary DNAs (cDNAs) and gene which code for an epithelial tumor antigen (H23-ETA), aberrantly expressed in human breast tumor tissue, are described here. A diversity of H23-ETA protein forms, is generated by a series of alternative splicing events that occur in regions located upstream and downstream to a central tandem 20 amino acid (aa) repeat array (TRA) that is rich in proline, serine and threonine residues. The upstream region shows that differential usage of alternative splice acceptor sites generates two protein forms containing putative signal peptides of varying hydrophobicities located at the NH2 terminus. The region downstream to the tandem repeat array indicates that one mRNA transcript is collinear with the gene and defines a 160 aa open reading frame (secreted or sec form). A second cDNA correlates with a mRNA that is generated by a series of splicing events and codes for 149 aa downstream to the TRA, identical with the aa sequence of the unspliced cDNA, after which it diverges and continues for an additional 179 aa. This sequence (transmembrane or tm form) contains a highly hydrophobic transmembrane domain of 28 aa followed by a hydrophilic “transfer-stop signal” (Arg Arg Lys) and a cytoplasmic domain of 72 aa. The various protein forms (alternative signal sequences, secreted and transmembrane) are likely routed to different cytoplasmic, cell membrane and extracellular compartments. Reverse PCR indicates that the relative ratios of the alternatively spliced forms vary in different epithelial tissues. To identify the individual protein species, monoclonal antibodies (mAb) are being generated against synthetic peptides unique to each form. The H23-ETA gene was also isolated and sequenced, demonstrating a putative promoter region that includes a ‘TATA’ box, Spl binding elements and an upstream putative hormone responsive element. Commensurate with these findings, H23-ETA expression was increased following hormonal treatment of BT549 breast tumor cells. These molecular studies have unravelled novel H23-ETA protein and gene structures, and facilitate future investigations that will focus on H23-ETA function and interaction with other cellular proteins.