A conserved spider silk domain acts as a molecular switch that controls fibre assembly

A huge variety of proteins are able to form fibrillar structures(1), especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand(2,3). Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal(4,5) and carboxy-terminal(6) domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation(7) initiated by changes in ionic composition(8,9) and mechanical stimuli known to align the repetitive sequence elements and promote beta-sheet formation(10-14). However, despite recent structural data(15), little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.

The role of salt and shear on the storage and assembly of spider silk proteins

Major ampullate silk fibers of orb web-weaving spiders have impressive mechanical properties due to the fact that the underlying proteins partially fold into helical/amorphous structures, yielding relatively elastic matrices that are toughened by anisotropic nanoparticulate inclusions (formed from stacks of beta-sheets of the same proteins). In vivo the transition from soluble protein to solid fibers involves a combination of chemical and mechanical stimuli (such as ion exchange, extraction of water and shear forces). Here we elucidate the effects of such stimuli on the in vitro aggregation of engineered and recombinantly produced major ampullate silk-like proteins (focusing on structure-function relationships with respect to their primary structures), and discuss their relevance to the storage and assembly of spider silk proteins in vivo. (C) 2009 Elsevier Inc. All rights reserved.

NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.

Spindle assembly checkpoint protein expression correlates with cellular proliferation and shorter time to recurrence in ovarian cancer.

Ovarian carcinoma (OC) is the most lethal of the gynecological malignancies, often presenting at an advanced stage. Treatment is hampered by high levels of drug resistance. The taxanes are microtubule stabilizing agents, used as first-line agents in the treatment of OC that exert their apoptotic effects through the spindle assembly checkpoint. BUB1-related protein kinase (BUBR1) and mitotic arrest deficient 2 (MAD2), essential spindle assembly checkpoint components, play a key role in response to taxanes. BUBR1, MAD2, and Ki-67 were assessed on an OC tissue microarray platform representing 72 OC tumors of varying histologic subtypes. Sixty-one of these patients received paclitaxel and platinum agents combined; 11 received platinum alone. Overall survival was available for all 72 patients, whereas recurrence-free survival (RFS) was available for 66 patients. Increased BUBR1 expression was seen in serous carcinomas, compared with other histologies (P = .03). Increased BUBR1 was significantly associated with tumors of advanced stage (P = .05). Increased MAD2 and BUBR1 expression also correlated with increased cellular proliferation (P < .0002 and P = .02, respectively). Reduced MAD2 nuclear intensity was associated with a shorter RFS (P = .03), in ovarian tumors of differing histologic subtype (n = 66). In this subgroup, for those women who received paclitaxel and platinum agents combined (n = 57), reduced MAD2 intensity also identified women with a shorter RFS (P < .007). For the entire cohort of patients, irrespective of histologic subtype or treatment, MAD2 nuclear intensity retained independent significance in a multivariate model, with tumors showing reduced nuclear MAD2 intensity identifying patients with a poorer RFS (P = .05).

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6 and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G(2)/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G(2) damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-d-manno-octulosonic acid and three glycero-manno-heptose residues. The HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.

Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

Polysaccharide co-polymerases:the enigmatic conductors of the O-antigen assembly orchestra

The O-antigen lipopolysaccharides on bacterial surface contain variable number of oligosaccharide repeat units with their length having a modal distribution specific to the bacterial strain. The polysaccharide length distribution is controlled by the proteins called polysaccharide co-polymerases (PCPs), which are embedded in the inner membrane in Gram-negative bacteria and form homo oligomers. The 3D structures of periplasmic domains of several PCPs have been determined and provided the first insights into the possible mechanism of polysaccharide length determination mechanism. Here we review the current knowledge of structure and function of these polysaccharide length regulators.

miR-433 overexpression attenuates the spindle assembly checkpoint response to paclitaxel

Paclitaxel is a microtubule inhibitory chemotherapeutic drug that is increasingly used for the treatment of solid tumours. In vitro studies have demonstrated that attenuating the spindle assemble checkpoint (SAC) alters the post-mitotic responses to paclitaxel. Furthermore, the aberrant expression of a number of the SAC proteins, MAD2, BUBR1, and Aurora A kinase, are associated with poor patient prognosis. We have identified a microRNA, miR-433, that regulates the expression of MAD2. Overexpression of miR-433 in Hela cells induced downregulation of MAD2 mRNA and protein expression. We have also shown that Hela cells overexpressing miR-433 and treated with paclitaxel are no longer capable of cyclin B stabilisation, and thus have lost the ability to activate the SAC in response to paclitaxel. In addition, cell viability assays showed that Hela cells overexpressing miR-433 and treated with paclitaxel have an attenuated response to paclitaxel compared with microRNA scrambled controls. We have characterised the levels of miR-433, MAD2 gene expression and MAD2 protein levels in a cohort of ovarian cancer cell lines. Cell viability assays on this cohort revealed that responsiveness to paclitaxel is associated with high MAD2 protein expression and lower miR-433 expression. We hypothesise that the expression of miR-433 when deregulated in cancer leads to altered MAD2 expression and a compromised SAC, a key feature underlying drug resistance to paclitaxel. In a pilot study of paired human breast tumour and normal breast tissue samples we have shown that expression levels of miR-433 are elevated in cancer tissue. Targeting this microRNA in cancer may improve the efficacy of paclitaxel in treating breast cancer and ovarian cancer.

Topological analysis of the Escherichia coli WcaJ protein reveals a new conserved configuration for the polyisoprenyl-phosphate hexose-1-phosphate transferase family

WcaJ is an Escherichia coli membrane enzyme catalysing the biosynthesis of undecaprenyl-diphosphate-glucose, the first step in the assembly of colanic acid exopolysaccharide. WcaJ belongs to a large family of polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) sharing a similar predicted topology consisting of an N-terminal domain containing four transmembrane helices (TMHs), a large central periplasmic loop, and a C-terminal domain containing the fifth TMH (TMH-V) and a cytosolic tail. However, the topology of PHPTs has not been experimentally validated. Here, we investigated the topology of WcaJ using a combination of LacZ/PhoA reporter fusions and sulfhydryl
labelling by PEGylation of novel cysteine residues introduced into a cysteine-less WcaJ. The results showed that the large central loop and the C-terminal tail both reside in the cytoplasm and are separated by TMH-V, which does not fully span the membrane, likely forming a "hairpin" structure. Modelling of TMH-V revealed that a highly conserved proline might contribute to a helix-break-helix structure in all PHPT members. Bioinformatic analyses show that all of these features are conserved in PHPT homologues from
Gram-negative and Gram-positive bacteria. Our data demonstrate a novel topological configuration for PHPTs, which is proposed as a signature for all members of this enzyme family

Interfacial self-assembly of a bacterial hydrophobin

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded β-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

DED or alive: assembly and regulation of the death effector domain complexes.

Death effector domains (DEDs) are protein-protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.