Protein buffering in model systems and in whole human saliva

The aim of this study was to quantify the buffer attributes (value, power, range and optimum) of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16%) between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate) staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s) belonging to the protein buffer system of human saliva.

Regulation of human GH receptor gene transcription by 20 and 22 kDa GH in a human hepatoma cell line.

The human GH gene is 1.7 kilobase pairs (kb) in length and is composed of five exons and four introns. This gene is expressed in the pituitary gland and encodes a 22 kDa protein. In addition to this predominant (75%) form, 5-10% of pituitary GH is present as a 20 kDa protein that has an amino acid (aa) sequence identical to the 22 kDa form except for a 15 aa internal deletion of residues 32-46 as a result of an alternative splicing event. Because it has been reported that non-22-kDa GH isoforms might be partly responsible for short stature and growth retardation in children, the aim of this study was to compare the impact of both 22 kDa and 20 kDa GH on GH receptor gene (GH receptor/GH binding protein (GHR/GHBP)) expression. Various concentrations of 20 kDa and 22 kDa GH (0, 2, 5, 12.5, 25, 50 and 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was measured by quantitative PCR. Addition of either 20 kDa or 22 kDa GH, at low or normal physiological concentrations (0, 2, 5, 12.5, 25 or 50 ng/ml) induced a dose-dependent increase in GHR/GHBP expression. However, a supraphysiological concentration of 20 kDa GH (150 ng/ml) resulted in a significantly lower (P<0.05) downregulation of GHR/GHBP gene transcription compared with the downregulation achieved by this concentration of 22 kDa GH. This difference might be explained by a decreased ability to form a 1 : 1 complex with GHR and/or GHBP, which normally occurs at high concentrations of GH. Nuclear run-on experiments and GHBP determinations confirmed the changes in GHR/GHBP mRNA levels. In conclusion, we report that both 20 kDa and 22 kDa GH, in low and normal physiological concentrations, have the same effect on regulation of GHR/GHBP gene transcription in a human hepatoma cell line. At a supraphysiological concentration of 150 ng/ml, however, 20 kDa GH has a less self-inhibitory effect than the 22 kDa form.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Their protein import systems are of similar complexity yet different composition than those in other eukaryotes. To elucidate the composition of the trypanosomal translocase of the inner mitochondrial membrane (TIM) we performed CoIPs of epitope-tagged TbTim17 and two other candidates in combination with SILAC-based quantitative mass spectrometry. This led to the identification of ten candidates for core TIM subunits. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a trypanosomatid-specific 42 kDa protein (Tim42) and two putative orthologues of inactive rhomboid proteases were analyzed further. All three proteins are essential in both life cycle stages and their ablation results in a strong protein import defect in vivo and in vitro. Blue native PAGE revealed their presence in a high molecular weight complex. Unlike anticipated, trypanosomes have a highly complex TIM translocase that has extensively been redesigned. None of the three novel TIM subunits has ever been associated with mitochondrial protein import. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. This suggests an exciting analogy between protein translocases of mitochondria and the ER.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER.

Growth hormone (GH)-releasing hormone increases the expression of the dominant-negative GH isoform in cases of isolated GH deficiency due to GH splice-site mutations

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Identification of a host cell target for the thiazolide class of broad-spectrum anti-parasitic drugs

The thiazolide nitazoxanide (NTZ) and some derivatives exhibit considerable in vitro activities against a broad range of parasites, including the apicomplexans Neospora caninum and Toxoplasma gondii tachyzoites. In order to identify potential molecular targets for this compound in both parasites, RM4847 was coupled to epoxy-agarose and affinity chromatography was performed. A protein of approximately 35 kDa was eluted upon RM4847-affinity-chromatography from extracts of N. caninum-infected human foreskin fibroblasts (HFF) and non-infected HFF, but no protein was eluted when affinity chromatography was performed with T. gondii or N. caninum tachyzoite extracts. Mass spectrometry analysis identified the 35 kDa protein as human quinone reductase NQO1 (P15559; QR). Within 8h after infection of HFF with N. caninum tachyzoites, QR transcript expression levels were notably increased, but no such increase was observed upon infection with T. gondii tachyzoites. Treatment of non-infected HFF with RM4847 did also lead to an increase of QR transcript levels. The enzymatic activity of 6-histidine-tagged recombinant QR (recQR) was assayed using menadione as a substrate. The thiazolides NTZ, tizoxanide and RM4847 inhibited recQR activity on menadione in a concentration-dependent manner. Moreover, a small residual reducing activity was observed when these thiazolides were offered as substrates.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Platelet glycoprotein Ia* is the processed form of multimerin--isolation and determination of N-terminal sequences of stored and released forms

Glycoprotein Ia* (GPIa*), a very high molecular mass, platelet alpha-granule protein consisting of 167 kDa subunits disulphide-linked in a multimeric structure, was first described by Bienz and Clemetson in 1989 (J. Biol. Chem. 264, 507-514). In 1991 Hayward et al. (J. Biol. Chem. 266, 7114-7120) independently identified a platelet protein with multimeric structure. Despite strong similarities to GPIa* they concluded that it was a novel multimeric protein and named it first p-155 and later, multimerin. Multimerin has also been found in endothelial cells and has been cloned recently from an endothelial cell cDNA library. This has made it possible for us to clarify the relationship between GPIa* and multimerin. GPIa* was isolated from platelet releasate and the N-terminal sequence of 167 kDa and 155 kDa subunit species were determined. The N-terminal 15 amino acids of GPIa* were identical to the deduced amino acids 184-198 of endothelial multimerin. The N-terminal sequence of the 155 kDa protein was identical to the deduced amino acids 318-326 of multimerin. Thus, platelet GPIa* (167 kDa) is the main processed form of multimerin stored in platelet alpha-granules. The GPIa*/processed multimerin (167 kDa) still contains an RGDS sequence near its N-terminus as well as an EGF domain which may be involved in binding to the platelet surface after release. This sequence and domain are cleaved off in the p-155 form, described earlier as platelet multimerin, which is probably formed after release from alpha-granules.

Bilinexin, a snake C-type lectin from Agkistrodon bilineatus venom agglutinates platelets via GPIb and alpha2beta1

A new snake protein, named bilinexin, has been purified from Agkistrodon bilineatus venom by ion-exchange chromatography and gel filtration chromatography. Under non-reducing conditions it has a mass of 110 kDa protein on SDS-PAGE. On reduction, it can be separated into five subunits with masses in the range 13-25 kDa. The N-terminal sequences of these subunits are very similar to those of convulxin or the alboaggregins, identifying bilinexin as a new member of the snake C-type lectin family, unusual in having multiple subunits. Bilinexin agglutinates fixed platelets. washed platelets and platelet rich plasma (PRP) without obvious activation (shape change) as confirmed by light microscope examination. Both inhibitory and binding studies indicate that antibodies against alpha2beta1 inhibit not only platelet agglutination induced by bilinexin, but also bilinexin binding to platelets. VM16d, a monoclonal anti-GPIbalpha antibody, completely inhibits platelet agglutination induced by bilinexin, and polyclonal antibodies against GPIbalpha prevent its binding to platelets. However, neither convulxin, polyclonal anti-GPVI antibodies, nor GPIIb/IIIa inhibitors affect its binding to and agglutination of platelets. Bilinexin neither activates GPIIb/IIIa integrin on platelets nor induces tyrosine phosphorylation of platelet proteins, nor increases intracellular Ca2+ in platelets. Like alboaggregin B, bilinexin agglutinates platelets, which makes it a good tool to investigate the differences in mechanism between snake C-type lectins causing platelet agglutination and those that induce full activation.

Mechano-regulated tenascin-C orchestrates muscle repair

Tenascin-C (TNC) is a mechano-regulated, morphogenic, extracellular matrix protein that is associated with tissue remodeling. The physiological role of TNC remains unclear because transgenic mice engineered for a TNC deficiency, via a defect in TNC secretion, show no major pathologies. We hypothesized that TNC-deficient mice would demonstrate defects in the repair of damaged leg muscles, which would be of functional significance because this tissue is subjected to frequent cycles of mechanical damage and regeneration. TNC-deficient mice demonstrated a blunted expression of the large TNC isoform and a selective atrophy of fast-muscle fibers associated with a defective, fast myogenic expression response to a damaging mechanical challenge. Transcript profiling mapped a set of de-adhesion, angiogenesis, and wound healing regulators as TNC expression targets in striated muscle. Expression of these regulators correlated with the residual expression of a damage-related 200-kDa protein, which resembled the small TNC isoform. Somatic knockin of TNC in fast-muscle fibers confirmed the activation of a complex expression program of interstitial and slow myofiber repair by myofiber-derived TNC. The results presented here show that a TNC-orchestrated molecular pathway integrates muscle repair into the load-dependent control of the striated muscle phenotype.

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Maintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate-limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knock-down results in loss of PG and a reduction of another mitochondria-specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin-isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co-migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.

Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.