Common structural traits for cystine knot domain of the TGFβ superfamily of proteins and three-fingered ectodomain of their cellular receptors

The transforming growth factor-β (TGFβ) superfamily of proteins and their receptors are crucial developmental factors for all metazoan organisms. Cystine-knot (CK) motif is a spatial feature of the TGFβ superfamily of proteins whereas the extra-cellular domains (ectodomains) of their respective receptors form three-fingered protein domain (TFPD), both stabilized by tight cystine networks. Analyses of multiple sequence alignments of these two domains encoded in various genomes revealed that the cystines forming the CK and TFPD folds are conserved, whereas the remaining polypeptide patches are diversified. Orthologues of the human TGFβs and their respective receptors expressed in diverse vertebrates retain high sequence conservation. Examination of 3D structures of various TGFβ factors bound to their receptors have revealed that the CK and TFPD domains display several similar spatial traits suggesting that these two different protein folds might have been acquired from a common ancestor.     

A complex of Shc and Ran-GTPase localises to the cell nucleus

The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 20 October 2008; received after revision 04 December 2008; accepted 15 December 2008     

A sperm GPI-anchored protein elicits sperm-cumulus cross-talk leading to the acrosome reaction

The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 11 August 2008; received after revision 18 December 2008; accepted 22 December 2008     

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotinconjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 September 2008; received after revision 17 December 2008; accepted 06 January 2009     

Loss of FrmA leads to increased cell-cell adhesion and impaired multi-cellular development of Dictyostelium cells

Cell-cell adhesion is a critical property of all multi-cellular organisms and its correct regulation is critical during development, differentiation, tissue building and maintenance, and many immune responses. The multi-talin-like FERM domain containing protein, FrmA, is required during starvation-induced multi-cellular development of Dictyostelium cells. Loss of FrmA leads to increased cell-cell adhesion and results in impaired multi-cellular development, slug migration and fruiting bodies. Further, mixing experiments show that FrmA null cells are excluded from the apex of wild-type mounds, to which cells that normally form the organising centre known as the tip sort. These data suggest a critical role for FrmA in regulating cell-cell adhesion, multi-cellular development and, in particular, the formation of the organising centre known as the tip. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 28 August 2008; received after revision 10 October 2008; accepted 21 October 2008     

Hydroxylation of demethoxy-Q6 constitutes a control point in yeast coenzyme Q6 biosynthesis

Coenzyme Q is a lipid molecule required for respiration and antioxidant protection. Q biosynthesis in Saccharomyces cerevisiae requires nine proteins (Coq1p–Coq9p). We demonstrate in this study that Q levels are modulated during growth by its conversion from demethoxy-Q (DMQ), a late intermediate. Similar conversion was produced when cells were subjected to oxidative stress conditions. Changes in Q₆/DMQ₆ ratio were accompanied by changes in COQ7 gene mRNA levels encoding the protein responsible for the DMQ hydroxylation, the penultimate step in Q biosynthesis pathway. Yeast coq null mutant failed to accumulate any Q late biosynthetic intermediate. However, in coq7 mutants the addition of exogenous Q produces the DMQ synthesis. Similar effect was produced by over-expressing ABC1/COQ8. These results support the existence of a biosynthetic complex that allows the DMQ₆ accumulation and suggest that Coq7p is a control point for the Q biosynthesis regulation in yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 04 September 2008; received after revision 22 October 2008; accepted 23 October 2008     

Dissection of a novel molecular determinant mediating Golgi to trans-Golgi network transition

Two major functions of the Golgi apparatus (GA) are formation of complex glycans and sorting of proteins destined for various subcellular compartments or secretion. To fulfill these tasks proper localization of the accessory proteins within the different sub-compartments of the GA is crucial. Here we investigate structural determinants mediating transition of the two glycosyltransferases β-1,4- galactosyltransferase 1 (gal-T1) and the α-1,3-fucosyltransferase 6 (fuc-T6) from the trans-Golgi cisterna to the trans-Golgi network (TGN). Upon treatment with the ionophore monensin both glycosyltransferases are found in TGN-derived swollen vesicles, as determined by confocal fluorescence microscopy and density gradient fractionation. Both enzymes carry a signal consisting of the amino acids E₅P₆ in gal-T1 and D₂P₃ in fuc-T6 necessary for the transition of these glycosyltransferases from the trans-Golgi cisterna to the TGN, but not for their steady state localization in the trans-Golgi cisterna. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 30 July 2008; received after revision 17 September 2008; accepted 29 September 2008     

For whom the bell tolls? DING proteins in health and disease

DING proteins, identified mainly by their eponymous N-terminal sequences, are ubiquitous in living organisms. Amongst bacteria, they are common in pseudomonads, and have been characterised with respect to genetics and structure. They form part of a wider family of phosphate-binding proteins, with emerging roles in phosphate acquisition and pathogenicity. Many DING proteins have been isolated in eukaryotes, in which they have been associated with very diverse biological activities, often in the context of possible signalling roles. Disease states in which DING proteins have been implicated include rheumatoid arthritis, lithiasis, atherosclerosis, some tumours and tumour-associated cachexia, and bacterial and viral adherence. Complete genetic and structural characterisation of eukaryotic DING genes and proteins is still lacking, though the phosphate-binding site seems to be conserved. Whether as bacterial proteins related to bacterial pathogenicity, or as eukaryotic components of biochemical signalling systems, DING proteins require further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure-function relationships in methionine adenosyltransferases

Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified. Received 22 August 2008; received after revision 22 September 2008; accepted 26 September 2008     

RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in Candida albicans

The calcineurin pathway has been reported to be essential for the development of azole resistance in Candida albicans. The depletion or ectopic over-expression of RTA2 increased or decreased susceptibility of C. albicans to azoles, respectively. CaCl₂- induced activation of the calcineurin pathway in wildtype C. albicans promoted resistance to azoles, while the Ca ²⁺ chelator (EGTA), calcineurin inhibitors (FK506 and cyclosporin A) and the deletion of RTA2 blocked the resistance-promoting effects of CaCl₂. Furthermore, we found that RTA2 was up-regulated in a calcineurin-dependent manner. The depletion of RTA2 also made the cell membrane of C. albicans liable to be destroyed by azoles and RTA2 over-expression attenuated the destroying effects. Finally, the disruption of RTA2 caused an increased accumulation of dihydrosphingosine (DHS), one of the two sphingolipid long-chain bases, by decreasing release of DHS. In conclusion, our findings suggest that RTA2 is involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 14 July 2008; received after revision 29 August 2008; accepted 16 September 2008     

Crystallin proteins and amyloid fibrils

Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, α-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer’s and Parkinson’s). In this review, the literature on the interaction of αB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials. Received 13 June 2008; received after revision 29 July 2008; accepted 05 August 2008     

The many faces of semaphorins: from development to pathology

The semaphorin family is a large group of proteins controlling cell migration and axonal growth cone guidance. These proteins are bi-functional signals capable of growth promotion or growth inhibition. Initially described in the nervous system, the majority of studies related to semaphorins and semaphorin signalling are nowadays performed in model systems outside the nervous system. Here, we provide an exhaustive review of the many faces of semaphorins both during developmental, regulatory and pathological processes. Indeed, because of their crucial fundamental roles, the semaphorins and their receptors represent important targets for the development of drugs directed at a variety of diseases. Received 22 August 2008; received after revision 22 September 2008; accepted 24 September 2008 L. Roth, E. Koncina, S. Satkauskas: These authors contributed equally to this work.     

Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases

A number of human diseases have been shown to be associated with mutation in the genes encoding leucine-rich-repeat (LRR)-containing proteins. They include 16 different LRR proteins. Mutations of these proteins are associated with 19 human diseases. The mutations occur frequently within the LRR domains as well as their neighboring domains, including cysteine clusters. Here, based on the sequence analysis of the LRR domains and the known structure of LRR proteins, we describe some features of different sequence variants and discuss their adverse effects. The mutations in the cysteine clusters, which preclude the formation of sulfide bridges or lead to a wrong paring of cysteines in extracellular proteins or extracellular domains, occur with high frequency. In contrast, missense mutations at some specific positions in LRRs are very rare or are not observed at all. Received 4 May 2005; received after revision 18 August 2005; accepted 1 September 2005     

Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway

The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca²⁺]_i ), and modulation of [Ca²⁺]_i via treatment with IP ₃R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca²⁺]_i, followed by Ca²⁺-ERK and Ca²⁺-mitochondria-caspase signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 05 July 2008; received after revision 25 August 2008; accepted 17 September 2008     

Welcome the Family of FANCJ-like Helicases to the Block of Genome Stability Maintenance Proteins

The FANCJ family of DNA helicases is emerging as an important group of proteins for the prevention of human disease, cancer, and chromosomal instability. FANCJ was identified by its association with breast cancer, and is implicated in Fanconi Anemia. Proteins with sequence similarity to FANCJ are important for maintenance of genomic stability. Mutations in genes encoding proteins related to FANCJ, designated ChlR1 in human and Chl1p in yeast, result in sister chromatid cohesion defects. Nematodes mutated in dog-1 show germline as well as somatic deletions in genes containing guanine-rich DNA. Rtel knockout mice are embryonic lethal, and embryonic stem cells show telomere loss and chromosomal instability. FANCJ also shares sequence similarity with human XPD and yeast RAD3 helicases required for nucleotide excision repair. The recently solved structure of XPD has provided new insight to the helicase core and accessory domains of sequence related Superfamily 2 helicases. The functions and roles of members of the FANCJ-like helicase family will be discussed. Received 17 September 2008; received after revision 24 October 2008; accepted 28 October 2008     

Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway

Advanced glycation end products (AGEs) play an important role in collagen deposition in diabetic cardiomyopathy. TRB3, a mammalian homolog of Drosophila tribbles, functions to increase glucose intolerance and regulates cell proliferation. We demonstrated that AGEs induce collagen type I expression but inhibit collagen type III expression, accompanied by increased TRB3 expression. Furthermore, the collagen type I induced byAGEs was down-regulated after inhibition of ERK and p38-MAPK, the collagen type III reduced by AGEs was up-regulated after inhibition of ERK. The expression of collagen types I and III regulated by AGEs through MAPK was partly reversed after treatment with TRB3 siRNA. It suggests that the TRB3/MAPK signaling pathway participates in the regulation of collagen types I and III by AGEs and may provide new therapeutic strategies for diabetic cardiomyopathy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 08 May 2008; received after revision 25 June 2008; accepted 22 July 2008 M. Tang, M. Zhong: These two authors contributed equally to this work.     

Light-harvesting complexes of vascular plants

Light-harvesting complexes (LHCs) located in the thylakoid membrane of plant chloroplasts are the collectors of solar radiation that fuel photosynthesis, and thus enable life on our planet. They consist of pigments that are non-covalently bound to light-harvesting proteins (Lhc proteins), which form a family whose members share a significant sequence identity. Due to their central role in photosynthesis, LHCs belong in several respects to the best-analysed membrane proteins. In the past decade, tremendous progress has been made in identifying new members of the Lhc family, in localising the LHCs within the photosystems, and in elucidating the structure and function of LHCs, which is summarised in this review. By contrast, gaining insight into the assembly process and the degradation of the LHCs could not keep pace. Therefore, topics for the next decade will be the elucidation of the location(s) and the operating mode of steps in the assembly and degradation process. Received 15 June 2008; received after revision 1 July 2008; accepted 10 July 2008