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Mechanisms of transcriptional regulation for the rat carbamyl phosphate synthetase I gene
No full textThe proximal promoter of the rat carbamyl phosphate synthetase I gene contains 4 cis-acting regulatory regions, designated sites GAC, I, II, and III. Transient transfection assays revealed that the GAG element functions as the main activator element, whereas sites I and III bind repressor proteins and site II binds an anti-repressor. We propose that sites I and III repress the CPS I promoter either by quenching the sole activator(s) bound to the GAG element, or by directly repressing the basal transcription apparatus. Footprint analyses of the promoter with and without a mutation within site II suggested that the site II-anti-repressor may interfere with the site I and site III repressors by a similar quenching mechanism. Although the elements within the proximal promoter exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity when analyzed either by in vitro transcription employing nuclear extracts from tissues that do not express the endogenous gene (i.e. brain, kidney, and spleen), or by transient transfection assays in non-CPS I-expressing cells (i.e. Rat1 and C6). However, we identified a cell-type specific enhancer that lies 10 kb upstream of the promoter. Unexpectedly, the enhancer functions as well in a gene-specific manner because enhancer-stimulated promoter activity was exclusively dependent on the GAG element within the CPS I proximal promoter. As a result, the GAG element is required to couple enhancer activity to the proximal promoter and exemplifies an efficient method whereby enhancers can be constrained to activate only their cognate promoters
Protein import into and across the mitochondrial outer membrane
No full textProtein import into the mitochondria is a result of a series of sequential binding interactions between a mitochondrial targeting signal and the translocation machinery of both mitochondrial membranes. The targeting signals contained within protein of the outer membrane are distinct from those which target proteins to other subcompartments. The transmembrane domain of the yeast outer membrane receptor protein yTom70 is capable of both targeting and inserting the protein into the outer membrane. The efficiency of this process is increased by the addition of a positively-charged region preceding the transmembrane region. These two structural domains co-operate to form a signal-anchor sequence selective for the outer membrane, since this is the first membrane encountered by the targeting signal.Consistent with this model, the signal-anchor sequence of the outer membrane protein yTom70 is also capable of importing into the inner membrane of mitochondria when the outer membrane is selectively removed. Import into the inner membrane requires the presence of an electrochemical potential across the lipid bilayer. Import proceeds in the absence of only when constructs are used which lack the positively-charged amino terminal region of the signal-anchor sequence. These results suggest that the positively-charged presequence leads the transmembrane domain into the import machinery and that is required to clear this region in order that the distal transmembrane region can enter the translocation pathway.The charged N-terminal 10 residues of yTom70 are incapable of directing import into intact mammalian mitochondria, however, are able to efficiently direct import into the matrix of yeast mitochondria or mammalian mitoplasts. This potentially cryptic signal is excluded from intact mammalian mitochondria due to the presence of the receptor protein Tom20, since replacement of yeast Tom20 with mammalian Tom20 confers the mammalian phenotype onto yeast. These results suggest that receptor proteins may also have the ability to screen potentially cryptic signals from distal components of the outer and inner membrane translocation machinery
Characterization of murine BH3-only BLK
No full textBLK has 43% sequence homology to BIK and an identical BH3 domain, a region that is critical for its proapoptotic activity, making it the strongest candidate mouse ortholog. In addition, the gene structures of these two proteins are similar in functional organization. BLK and BIK activate apoptosis in similar fashion in both human and mouse cell lines, dependent on the presence of a functional BH3 domain and active caspases. BLK, like BIK, is very unstable and can be stabilized by addition of pan caspase inhibitor, zVAD-fmk. In contrast to BIK, BLK is not upregulated by p53 at the RNA or protein level in several mouse cell line systems. Furthermore, BLK localizes to the mitochondria, not to the endoplasmic reticulum where BIK resides. To further investigate the localization of BLK and BIK, the transmembrane domains of these proteins were swapped. BLKBIK and BIKBLK both localized to the endoplasmic reticulum. Enhanced Green Fluorescent Protein fused to the transmembrane domains of BLK and BIK also localized to the endoplasmic reticulum. The mitochondrial localization of BLK despite its transmembrane domain preference for the endoplasmic reticulum suggests that the N-terminal portion of BLK plays a role in targeting. Collectively, these results argue that BLK is not the functional mouse homolog of BIK
Assembly pathways of outer mitochondrial membrane proteins
No full textThe pathway of membrane insertion and assembly of a signal anchor sequence specific for the outer mitochondrial membrane has been investigated. Signal anchor protein insertion into the outer membrane, in vitro, was found to overlap with a general import pathway followed by the outer membrane protein, porin, as well as matrix-targeted proteins. However, signal anchor protein insertion did not require a postreceptor import step involved in porin insertion and matrix protein translocation. Also, in contrast to the membrane insertion of porin, signal anchor protein insertion did not require nucleoside triphosphates for transfer of bound precursor to the membrane assembled form. Following outer membrane integration, a hybrid protein containing the signal anchor sequence of yeast Tom70 was found to assemble into homodimers. Dimerization was mediated by the transmembrane domain. A sequence motif containing alanine residues clustered on one face of the predicted membrane-spanning -helix was important for dimerization, perhaps allowing favourable close packing of the transmembrane helices
The anti-apoptotic function of adenovirus E1B 19kDa protein /
No full textE1B 19kDa protein (19K), a member of Bcl-2 family of protein, is an adenovirus early gene product. It functions to suppress apoptosis of the host cell caused by viral infection. The exact role of E1B 19kDa in regulating apoptosis in response to many different death stimuli has not been investigated.We found that hygromycin B, an aminoglycoside antibiotic that is used to establish stable mammalian cell lines, kill cells by apoptosis. Apoptosis induced by Hygromycin B is p53-independent and can be blocked both by E1B 19kDa and Bcl-2, showing that E1B 19kDa, like Bcl-2, may confer protection against apoptosis in a variety of circumstances.The sequence similarities between E1B 19kDa and Bcl-2 are largely restricted to the BH1 and BH3 domains. Our sequence alignment analysis shows that E1B 19kDa also contains a BH4 region overlapping its BH3 domain. We find that the BH1 domain of E1B 19kDa contributes to its anti-apoptotic function and its ability to interact with Bax. A conserved residue Gly 87 in the BH1 domain of E1B 19kDa that is critical for Bcl-2 and Bax interaction is also important for the E1B 19kDa and Bax interaction. We do not detect an interaction between E1B 19kDa and Bad, suggesting that E1B 19kDa is a structural and functional homolog of Bcl-2 but also has its unique features.Bap31, a 28kDa polytoptic integral protein of the endoplasmic reticulum, is part of an ER protein complex that also includes Bcl-2/Bcl-xL, procaspase-8, and a CED-4-like protein. E1B 19kDa does not interact with Bap31; nor does it interact with Bcl-2/Bcl-xL. We have made a set of chimeric E1B 19kDa proteins carrying different BH domains from Bcl-2 to test their protein-protein interaction properties. We show that the BH3 domain of Bcl-2, when substituted for the homologous region of E1B 19kDa, confers the properties of interaction with Bap31 and Bcl-xL onto 19K. The subcellular distribution and antiapoptotic function of the 19K/Bcl-2BH3 is very similar to that of wild type 19K. These results show that the BH3 domain of Bcl-2/Bcl-xL has an important role in mediating Bcl-2/Bcl-xL and Bap31 interaction
Functional expression of the human mitochondrial import receptor Tom20 in escherichia coli
No full textThe work described herein represents a preliminary step in the characterisation of the human Tom20 protein and its role in the mammalian mitochondrial import machinery. A hexahistidine-tagged form of the human homologue of Tom20 has been over-expressed in Escherichia coli and purified under native conditions. This Tom20 protein has been shown, in these studies, to compete for the import of a matrix-destined mitochondrial precursor protein, pre-ornithine carbamoyltransferase (pOCT), into mitochondria isolated from a mammalian source (rat heart), as well as to directly bind the presequence-bearing pOCT in vitro. Furthermore, a protocol has been established for the isolation of import-competent outer membrane vesicles from rat liver mitochondria. These vesicles have been preliminarily characterised in terms of the import of two outer membrane-destined DHFR fusion proteins, Tom70(1-29)-DHFR and Tom20(1-28)-DHFR, and have been shown to exhibit similar characteristics/requirements for protein import as seen for intact mitochondria: temperature sensitivity, the dependence upon cytosolic ATP, and the requirement for protease-sensitive outer membrane surface components
Regulation of cytochrome c release from mitochondria during apoptosis by BCL-2 family members
No full textApoptosis is a stringently regulated programmed cell death pathway by which a cell will kill itself in response to extensive damage, stress or a targeted signal. It is an important process in development, which requires the elimination of superfluous cells, as well as the regulation of mitotically active cells in a fully developed organism. The key regulators at the heart of the apoptotic pathway are a group of proteins known as the BCL-2 family, containing both pro- and anti-apoptotic members. These proteins are intimately involved in mitochondrial and endoplasmic reticulum dysfunction triggered by an apoptotic signal. These dysfunctions include release of cytochrome c from the mitochondria and the efflux of calcium from the ER. It is the BCL-2 family of proteins, their function and regulation, which is the focus of this thesis. We demonstrate that BID, a member of the pro-apoptotic BH3-only subfamily, when processed to its active form, tBID, will induce the integration of another pro-apoptotic protein, BAX, into the outer mitochondrial membrane which leads to the release of cytochrome c from the inter membrane space. There are, however, both BID-dependent and BID-independent pathways that are responsible for the insertion of BAX into the mitochondrial membrane though in either pathway the release of cytochrome c remains dependent on tBID. tBID can also induce the oligomerization of BAK, another pro-apoptotic member constitutively integrated in the outer membrane of the mitochondria and closely related to BAX, which will also lead to cytochrome c release. This oligomerization is a two-step mechanism, first tBID will interact with and activate BAK by inducing a change in conformation of its NH2-terminus, then the activated form of BAK will initiate an auto-oligomerization cascade with other inactive BAK molecule. Anti-apoptotic BCL-2 will inhibit the auto-oligomerization of BAK and subsequent release of cytochrome c by binding the active
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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