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Ubiquitin-activating enzyme (E1) is the first enzyme of the ubiquitination pathway and is required to activate ubiquitin. E1 of Saccharomyces cerevisiae is a large multidomain monomeric protein. There are no studies available on the domains of yeast E1 as independent entities. Four domains of E1 namely, first catalytic cysteine half-domain (FCCH), four-helix bundle (4HB), second catalytic cysteine half-domain (SCCH) and ubiquitin fold domain (UFD) were characterised to understand their structural and functional independence vis-a-vis full length E1. Spectroscopic characterisation using circular dichroism and fluorescence suggested that these domains can act as independent folding units and attain native-like secondary structure. The structural features obtained with the peptides SCCH and FCCH of S. cerevisiae bear a high degree of structural similarity to the corresponding fragments of mouse from literature. Nearly 50% of the residues of the 4HB domain of the S. cerevisiae sample showed helical conformation. They displayed a high degree of conservation when compared with 4HB of mouse with respect to their identity and arrangement. The fragment UFD of yeast formed an α/β domain as in the whole protein and exhibited 45% homology with that of mouse, showing a similar arrangement of α and β elements in its secondary structure. Overexpression of the domains in vivo indicated that the SCCH domain and to some extent UFD apparently interfere with cellular functions such as survival under various stresses. [ABSTRACT FROM AUTHOR]

Ubiquitination is tightly regulated to control degradation, localization and function of various proteins. Ubiquitination is catalysed by three enzymes, namely E1, E2 and E3. The specificity shown by E2s for E3s holds key to regulation of ubiquitination. Here we focussed on the E2 enzymes, UBC4 and UBC5 of Saccharomyces cerevisiae, which are almost identical differing only by 11 residues. They show functional complementation in protein degradation, especially during stress response. Existence of two almost identical proteins suggests specialized requirement of one of them under selective conditions. To understand the reasons for the residue differences between them, mutations were introduced in the UBC4 gene to generate single residue variants by swapping with codons from UBC5. Though the variants are found to be functionally active in Δubc4Δubc5 strain of yeast, they cause reduced growth under normal conditions, altered survival under heat and antibiotic stresses, when compared with UBC4. The variants indicated decrease in protein stability theoretically. Hence, the residues of UBC5 individually do not confer any structural advantage to UBC4. Interactive proteins of UBC4 are nearly three times more than those of UBC5. UBC5, therefore, is a functionally minimized version, evolved as another means of regulation to meet cell stage specific needs. [ABSTRACT FROM AUTHOR]

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/β domain consisting of α/β barrel domain (α/β) preceded by an overhang of 50 residues at its N-terminus (N50) and β sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/β domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/β of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/β and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/β and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/β and CmODC can be employed to study the function of novel proteins through their rapid removal. [ABSTRACT FROM AUTHOR]

Studies on knockout mutants and conditional mutants are invaluable to biological research and have been used extensively to probe the intricacies of biological systems through loss of function associated with attenuation of a particular protein. Besides, RNAi technology has been developed in recent years to further aid the process of scientific inquiry. Even though, the methods, dealing with DNA and RNA have met with great success, are not without their shortcomings. In order to overcome the inadequacies of existing methods, a host of new techniques, aimed at knockdowns at the protein rather than the nucleic acid level, have been devised. Essentially, these methods can achieve rapid degradation of cellular pools of a target protein in response to an inducible signal coupled with dose-dependent modulation and exquisite temporal control, features which are absent from techniques involving manipulations at the DNA or RNA level. This review aims to provide a broad overview of a gamut of these methods, while highlighting the strengths and weaknesses of each one. Last two decades of advances presented here in the field of targeted protein degradation serve as a beacon to further research and are likely to find applications in the areas of medicine and allied fields of biology. [ABSTRACT FROM AUTHOR]

Ubiquitin is a small, globular protein, structure of which has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 β-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications. [ABSTRACT FROM AUTHOR]

Using the subjects desalination and educational psychology, the scatter of periodical articles over periodical titles was compared at two levels, the second level being a random sample of periodical articles cited by the first level. Several measures were used to compare the extent of scatter at the two levels. Some methods commonly used in bibliometrics produced conflicting evidence on whether the citing literature (first-level) or the cited (second-level) was more scattered. A computer-intensive sampling procedure, known as the Bootstrap method, was then used to estimate the scatter of the total cited population from the scatter of the empirical sample. Cumulative distributions were prepared to show what percentage of periodicals accounted for various percentages of articles at each level of scatter. Only at the 90th percentile of articles did the percentage of periodical titles in the cited literature significantly exceed that of the citing literature. At the tail-end of the Bradford-type distribution, the cited literature appears to be more scattered than the literature citing it. [ABSTRACT FROM AUTHOR]