ABSTRACT
Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter. CONCLUSION: Short SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT.
Subject(s)
alpha 1-Antitrypsin , Humans , Mutation , Open Reading Frames/genetics , RNA, Messenger/genetics , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolism , alpha 1-Antitrypsin Deficiency/geneticsABSTRACT
Troponin complex is a component of skeletal and cardiac muscle thin filaments. It consists of three subunits - troponin I, T, and C, and it plays a crucial role in muscle activity, connecting changes in intracellular Ca2+ concentration with generation of contraction. In spite of more than 40 years of studies, many aspects of troponin functioning are still not completely understood, and several models describing the mechanism of muscle contraction exist. Being a key factor in the regulation of cardiac muscle contraction, troponin complex is utilized in medicine as a target for some cardiotonic drugs used in the treatment of heart failure. A number of mutations in troponin subunits are associated with development of different types of cardiomyopathy. Moreover, for the last 25 years cardiac isoforms of troponin I and T have been widely used for immunochemical diagnostics of pathologies associated with cardiomyocyte death (myocardial infarction, myocardial trauma, and others). This review summarizes the existing evidence on the structure and function of troponin complex subunits, their role in the regulation of cardiac muscle contraction, and their clinical applications.
Subject(s)
Heart Diseases/metabolism , Myocardium/metabolism , Troponin C/chemistry , Troponin C/metabolism , Troponin I/chemistry , Troponin I/metabolism , Troponin T/chemistry , Troponin T/metabolism , Animals , Heart Diseases/genetics , Humans , Troponin C/genetics , Troponin I/genetics , Troponin T/geneticsABSTRACT
The cardiac isoform of troponin I is a reliable biomarker of damaged cardiomyocytes that accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibody 19C7 recognizes troponin I in the bloodstream with high affinity and specificity. Recombinant antibodies can be used to improve detection systems based on monoclonal antibodies produced with hybridoma technology. In the present study, we compare the properties of monoclonal antibody 19C7 and its recombinant fragments. It is shown that the recombinant antibody fragments demonstrate similar affinity values as monoclonal antibodies and can be applied for troponin I detection.