Mammalian proapoptotic factor ChaC1 and its homologues function as γ‐glutamyl cyclotransferases acting specifically on glutathione

ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as γ‐glutamyl cyclotransferases acting specifically to degrade glutathione but not other γ‐glutamyl peptides. The overexpression of these proteins (but not the catalytically dead E>Q mutants) led to glutathione depletion and enhanced apoptosis in yeast. The ChaC family is conversed across all phyla and represents a new pathway for glutathione degradation in living cells, and the first cytosolic pathway for glutathione degradation in mammalian cells.     

Binding determinants of the small heat shock protein, αB‐crystallin: recognition of the ‘IxI’ motif

Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate‐prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB‐crystallin (‘αB’), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α‐crystallin domain (ACD) forms dimeric building blocks, while flanking N‐ and C‐termini direct the formation of larger sHSP oligomers. The most commonly observed inter‐subunit interaction involves a highly conserved C‐terminal ‘IxI/V’ motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB‐ACD. IxI‐mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers.     

5α,6α-Epoxy-cholestan-3β-ol (cholesterol α-oxide): A specific substrate for rat liver glutathione transferase B

A semi-micro assay was developed for the conjugation of 5α,6α-epoxy-cholestan-3β-ol (cholesterol α-oxide) with glutathione. The soluble supernatant of rat liver homogenate catalysed the reaction at a rate of 0.2–0.5 pmol.min⁻¹ .mg protein⁻¹ with 4μM cholesterol α-oxide, while the reaction in the presence of GSH alone was barely detectable. Enzymic activity in the soluble supernatant was due equally to the two forms of glutathione transferase B (100 pmol.min⁻.mg protein⁻¹), glutathione transferases AA, A, C and E being unreactive. The activity of purified glutathione transferase B was about 5-times that expected from the activity of the soluble supernatant. Complex enzyme kinetics were obtained suggestive of substrate inhibition.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Electron microscopic demonstration of a common structural motif in human complement factor C3 and rat α1 -inhibitor 3 (murinoglobulin)

Electron microscopy of two homologous giant proteins revealed that complement factor C3 and α_i-inhibitor 3 have a common structural motif of a semicircularly bent string 18–20 nm long with two or three bumps indicating globular domains. C3 had a structure similar to the letter C with a small but distinct hole in the center. α₁-Inhibitor 3 was a more complete ring sometimes ajar at one corner. When the latter was treated with a proteinase, it became slightly flattened and adopted a squarish C-shape.     

Dissecting α-helices: Position-specific analysis of α-helices in globular proteins

An analysis of the amino acid distributions at 15 positions, viz., N“, N′, Ncap, N1, N2, N3, N4, Mid, C4, C3, C2, C1, Ccap, C′, and C” in 1,131 α-helices reveals that each position has its own unique characteristics. In general, natural helix sequences optimize by identifying the residues to be avoided at a given position and minimizing the occurrence of these avoided residues rather than by maximizing the preferred residues at various positions. Ncap is most selective in its choice of residues, with six amino acids (S, D, T, N, G, and P) being preferred at this position and another 11 (V, I, F, A, K, L, Y, R, E, M, and Q) being strongly avoided. Ser, Asp, and Thr are all more preferred at Ncap position than Asn, whose role at helix N-terminus has been highlighted by earlier analyses. Furthermore, Asn is also found to be almost equally preferred at helix C-terminus and a novel structural motif is identified, involving a hydrogen bond formed by N^δ2 of Asn at Ccap or C1 position, with the backbone carbonyl oxygen four residues inside the helix. His also forms a similar motif at the C-terminus. Pro is the most avoided residue in the main body (N4 to C4 positions) and at C-ter-minus, including Ccap of an α-helix. In 1,131 α-helices, no helix contains Pro at C3 or C2 positions. However, Pro is highly favoured at N1 and C′. The doublet X-Pro, with Pro at C′ position and extended backbone conformation for the X residue at Ccap, appears to be a common structural motif for termination of α-helices, in addition to the Schellman motif. Main body of the helix shows a high preference for aliphatic residues Ala, Leu, Val, and Ile, while these are avoided at helix termini. A propensity scale for amino acids to occur in the middle of helices has been obtained. Comparison of this scale with several previously reported scales shows that this scale correlates best with the experimentally determined values. Proteins 31:460–476, 1998. © 1998 Wiley-Liss, Inc.     

Structure of an integrin with an αI domain,complement receptor type 4

We report the structure of an integrin with an αI domain, α_Xβ₂, the complement receptor type 4. It was earlier expected that a fixed orientation between the αI domain and the β‐propeller domain in which it is inserted would be required for allosteric signal transmission. However, the αI domain is highly flexible, enabling two βI domain conformational states to couple to three αI domain states, and greater accessibility for ligand recognition. Although α_Xβ₂ is bent similarly to integrins that lack αI domains, the terminal domains of the α‐ and β‐legs, calf‐2 and β‐tail, are oriented differently than in αI‐less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the β₂‐tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.     

Solution structure of protein synthesis initiation factor 1 from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa‐IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa‐IF1. Pa‐IF1 consists of a five‐stranded β‐sheet with an unusual extended β‐strand at the C‐terminus and one short α‐helix arranged in the sequential order β1‐β2‐β3‐α1‐β4‐β5. The structure adopts a typical β‐barrel fold and contains an oligomer‐binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α‐helix may compose the binding interface with the 30S subunit.     

Design of stable α‐helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ～70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The matrix protein CCN1/CYR61 is required for αVβ5‐mediated cancer cell migration

CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real‐time PCR to establish the expression profile of CYR61 and integrin α_Vβ₅ in three non–small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α_Vβ₅ integrin. Further, we investigated whether tumour cell–secreted CYR61 can facilitate cell migration by interacting with the α_Vβ₅ integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α_Vβ₅ expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α_vβ₅ integrin as proteins that cooperate to mediate cancer cell migration. Copyright © 2012 John Wiley & Sons, Ltd.     

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin‐1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)‐β and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF‐β‐induced expression of CTGF in fibroblasts by an interleukin (IL)‐1 α‐dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL‐1α and β. Human dermal fibroblasts and NIH 3T3 cells were treated with IL‐1α or β in presence or absence of TGF‐β1. IL‐1 suppressed basal and TGF‐β‐induced CTGF mRNA and protein expression. IL‐1α and β inhibited TGF‐β‐stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3‐binding CAGA elements. Furthermore, IL‐1α and β inhibited TGF‐β‐stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF‐β activated kinase1 (TAK1) is necessary for IL‐1 inhibition of TGF‐β‐stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226–1233, 2010. Published 2010 Wiley‐Liss, Inc.     

Identification of ciliary neurotrophic factor receptor α as a mediator of neurotoxicity induced by α‐synuclein

Accumulating evidence suggests that extracellular α‐synuclein (eSNCA) plays an important role in the pathogenesis of Parkinson's disease or related synucleinopathies by inducing neurotoxicity directly or indirectly via microglial or astroglial activation. However, the mechanisms by which this occurs remain to be characterized. To explore these mechanisms, we combined three biochemical techniques – stable isotope labeling of amino acid in cell cultures (SILAC), biotin labeling of plasma membrane proteins followed by affinity purification, and analysis of unique proteins binding to SNCA peptides on membrane arrays. The SILAC proteomic analysis identified 457 proteins, of which, 245 or 172 proteins belonged to membrane or membrane associated proteins, depending on the various bioinformatics tools used for interpretation. In dopamine neuronal cells treated with eSNCA, the levels of 86 membrane proteins were increased and 35 were decreased compared with untreated cells. In peptide array analysis, 127 proteins were identified as possibly interacting with eSNCA. Of those, seven proteins were overlapped with the membrane proteins that displayed alterations in relative abundance after eSNCA treatment. One was ciliary neurotrophic factor receptor, which appeared to modulate eSNCA‐mediated neurotoxicity via mechanisms related to JAK1/STAT3 signaling but independent of eSNCA endocytosis.     

Relationships between translation of pro α1(I) and pro α2(I) mRNAs during synthesis of the type I procollagen heterotrimer

Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro α chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro α1(I) and pro α2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro α-chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro α1 homotrimer or the normal heterotrimer. Recombinant human pro α1(I) and pro α2(I) C DNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate ± canine pancreatic microsomes. Cycloheximide (100 μg/ml) was added and the polysomes were collected and fractionated on a 15–50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing), W8 (pro α1(I) homotrimer), and A2′ (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro α1(I) and pro α2(I) messages proceeded independently in the cell-free, membrane-free systems, but were coordinately altered in the presence of membrane. The cell-free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro α chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding. © 1995 Wiley-Liss, Inc.