The transthyretin-related protein family

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretinrelated protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligandbinding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used doublestranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity

Promiscuous DNA synthesis by human DNA polymerase θ

The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3′-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks

Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild-type sea bream TTR (sbTTRWT) plus two mutants in which six (sbTTRM6) and twelve (sbTTRM12) N-terminal residues were removed. Ligandbinding studies revealed similar affinities for T3 (Kd=10.6±1.7nM) and T4 (Kd=9.8±0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3±15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine-T.In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR

Signal-sequence induced conformational changes in the signal recognition particle

Co-translational protein targeting is an essential, evolutionarily conserved pathway for delivering nascent proteins to the proper cellular membrane. In this pathway, the signal recognition particle (SRP) first recognizes the N-terminal signal sequence of nascent proteins and subsequently interacts with the SRP receptor. For this, signal sequence binding in the SRP54 M domain must be effectively communicated to the SRP54 NG domain that interacts with the receptor. Here we present the 2.9 angstrom crystal structure of unbound- and signal sequence bound SRP forms, both present in the asymmetric unit. The structures provide evidence for a coupled binding and folding mechanism in which signal sequence binding induces the concerted folding of the GM linker helix, the finger loop, and the C-terminal alpha helix alpha M6. This mechanism allows for a high degree of structural adaptability of the binding site and suggests how signal sequence binding in the M domain is coupled to repositioning of the NG domain

Heating of proteins as a means of improving crystallization: a successful case study on a highly amyloidogenic triple mutant of human transthyretin

By heating a highly amyloidogenic mutant of the human plasma protein transthyretin at 328 K for 48 h, diffraction-quality crystals could be reproducibly produced. The procedure precipitated ∼40% of the protein, but rendered what remained in solution more homogenous

Thyroid hormone binding by recombinant sea bream transthyretin: the role of the N-terminal region

Transthyretin (TTR) along with thyroxine-binding globulin (TBG) and albumin (ALB) constitute Thyroid Hormone-Binding Proteins (THBP) in vertebrates. These proteins bind and transport thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood. THBP are poorly characterized in fish and in the present study binding of THs by sea bream TTR was determined. Teleost TTR is a single polypeptide chain of 130 amino acids, but in common with mammals the functional form in the plasma is a tetramer. In contrast to mammalian TTR (127 amino acids), fish TTR has a longer N-terminal region and the latter has been proposed to influence TH binding. In the present study recombinant sea bream TTR (sbTTR, wild type), plus two recombinant N-terminal mutants were produced and purified. Binding of [I125]-T3 to the purified TTRs was confirmed by polyacrylamide gel electrophoresis under nondenaturing conditions followed by autoradiography and confirmed all bind THs as a tetramer. Ligand binding studies with labeled [I125]-T3 were performed and revealed that [I125]-T3 was displaced in a similar way from wild type and mutant TTRs by increasing concentrations of unlabeled T3. Similar Kd values were obtained for both T3 and T4 binding to wild type and mutant TTRs indicating that the N-terminal region does not seem to be important for the binding characteristics of sbTTR

Structural insights into SRP RNA: An induced fit mechanism for SRP assembly

Proper assembly of large protein–RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein–RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-Å resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs

Computerized tomography before the final treatment cycle of neoadjuvant chemotherapy or induction chemotherapy in muscle-invasive urinary bladder cancer, cannot predict pathoanatomical outcomes and does not reflect prognosis-results of a single centre retrospective prognostic study

Background: Evaluating the routine of using control computer tomography (cCT) for determining the response status of muscle-invasive bladder cancer (MIBC) prior to final cycle of neoadjuvant chemotherapy (NAC) or induction chemotherapy (IC), in terms of predicting histopathological pTNM-staging and pathoanatomical responses/non-responses. Secondly, predicting two and three-year overall survival (OS). Methods: Seventy-seven patients with localized MIBC (cT2-4aN0M0) and 3 patients with minimal nodal dissemination (cN1-2), undergoing NAC or IC and radical cystectomy (RC), the years 2006–2014 at Norrland university hospital in Umeå, Sweden. Baseline pre-cystectomy CTs and cCTs prior to final chemotherapy-cycle, were reviewed and underwent attempted RECIST-criteria categorization, into five response/non-response related subgroups (n=71). The diagnostic accuracy of cCT in comparison with pTNM was assessed using sensitivity, specificity, positive- and negative likelihood ratios. OS for 2 and 3 years was calculated, both in relation to histopathological pTNM-stages in all patients (n=80) and for the patients with cCT-evaluated categories (n=71). Multivariable analysis for OS, was performed in correlation to pTNM-stages firstly, and to radiological staging secondly. Results: The sensitivity of cCT to predict non-responders according to pTMN was 64% and specificity 36%. The positive likelihood ratio=1 and the negative likelihood ratio =1. CT-evaluations couldn’t accurately predict pTNM-stages in terms of response/non-response. No statistically significant results were found in correlating cCTs with two and three-year OS. Conclusions: cCT prior to planned final preoperative chemotherapy-cycle in MIBC patients undergoing NAC or IC, has a poor correlation with pTNM and cannot predict pathoanatomical responses. Prediction of OS based on cCTs is unfeasible. Keywords: Computed X ray tomography; cystectomy interdisciplinary health team; neoadjuvant therapy; urinary bladder neoplasm

Expression and purification of SfaXII, a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli

AbstractPathogenic Escherichia coli strains commonly harbor genes involved in formation of fimbriae, such as the sfaII fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. The sfaXII gene, located at the distal end of the sfaII operon, was recently shown to play a role in controlling virulence-related gene expression in extraintestinal pathogenic E. coli (ExPEC). Until now, detailed characterization of the SfaXII protein has been hampered by difficulties in obtaining large quantities of soluble protein. By a rational modeling approach, we engineered a Cys70Ser mutation, which successfully improved solubility of the protein. Here, we present the expression, purification, and initial characterization of the recombinant SfaXIIC70S mutant. The protein was produced in E. coli BL21 (DE3) cells grown in autoinduction culture media. The plasmid vector harbored DNA encoding the SfaXIIC70S protein N-terminally fused with a six histidine (H6) sequence followed by a ZZ tag (a derivative of the Staphylococcus protein A) (H6-ZZ tag). The H6-ZZ tag was cleaved off with Tobacco Etch Virus (TEV) protease and the 166 amino acid full-length homo-dimeric protein was purified using affinity and size-exclusion chromatography. Electrophoretic mobility gel shift assays and atomic force microscopy demonstrated that the protein possesses DNA-binding properties, suggesting that the transcriptional regulatory activity of SfaXII can be mediated via direct binding to DNA