The E4orf6/E1B55K E3 ubiquitin ligase complexes of human adenoviruses exhibit heterogeneity in composition and substrate specificity

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin α3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin α3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.     

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.     

Proteomic analyses of intermediate filaments reveals cytokeratin8 is highly acetylated--implications for colorectal epithelial homeostasis.

Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

CD106 Identifies a Subpopulation of Mesenchymal Stem Cells with Unique Immunomodulatory Properties

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106⁺cells and CD106⁻cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106⁺CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106⁺CV-MSCs expressed more cytokines than CD106⁻CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.     

Enhanced virulence effect of K1 polysaccharide in neonatal rats challenged withE. coli

Purified K1 polysaccharide enhanced the virulence of non-K1Escherichia coli species when given by orogastric feeding to neonatal rats. Neonatal rats developedE. coli bacteremia when K1 polysaccharide was given concomitantly with non-K1E. coli, whereasE. coli bacteremia did not develop when non-K1E. coli was given alone.E. coli K1 species did cause bacteremia and meningitis when fed to neonatal rats. The mechanism by which k1 polysaccharide enhances virulence can be studied with this model of bacteremia development in neonatal rats.