Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure.

α-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro Oxidative stress has been implicated in PD and oxidized α-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either α- or ß-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles.

BACKGROUND: Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known. METHODS: Three IDPs (α-casein, Sic1 and α-synuclein) and lysozyme are compared, describing conformational properties inside HC on silica NPs by circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy. RESULTS: IDPs inside HC are largely unstructured, but display small, protein-specific conformational changes. A minor increase in helical content is observed for α-casein and α-synuclein, reminiscent of membrane effects on α-synuclein. Frozen in their largely disordered conformation, bound proteins do not undergo folding induced by dehydration, as they do in their free forms. While HC thickness approaches the hydrodynamic diameter of the protein in solution for lysozyme, it is much below the respective values for IDPs. NPs boost α-synuclein aggregation kinetics in a dose-dependent manner. CONCLUSIONS: IDPs maintain structural disorder inside HC, experiencing minor, protein-specific, induced folding and stabilization against further conformational transitions, such as formation of intermolecular beta-sheets upon dehydration. The HC is formed by a single layer of protein molecules. SC likely plays a key role stabilizing amyloidogenic α-synuclein conformers. GENERAL SIGNIFICANCE: Protein-NP interactions can mimic those with macromolecular partners, allowing dissection of contributing factors by rational design of NP surfaces. Application of NPs in vivo should be carefully tested for amyloidogenic potential.

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein.

The intrinsically disordered and amyloidogenic protein α-synuclein (AS) has been linked to several neurodegenerative states, including Parkinson's disease. Here, nanoelectrospray-ionization mass spectrometry (nano-ESI-MS), ion mobility (IM), and native top-down electron transfer dissociation (ETD) techniques are employed to study AS interaction with small molecules known to modulate its aggregation, such as epigallocatechin-3-gallate (EGCG) and dopamine (DA). The complexes formed by the two ligands under identical conditions reveal peculiar differences. While EGCG engages AS in compact conformations, DA preferentially binds to the protein in partially extended conformations. The two ligands also have different effects on AS structure as assessed by IM, with EGCG leading to protein compaction and DA to its extension. Native top-down ETD on the protein-ligand complexes shows how the different observed modes of binding of the two ligands could be related to their known opposite effects on AS aggregation. The results also show that the protein can bind either ligand in the absence of any covalent modifications, such as oxidation.

Neuronal haemoglobin induces loss of dopaminergic neurons in mouse Substantia nigra, cognitive deficits and cleavage of endogenous α-synuclein.

Parkinson's disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. α-synuclein (α-syn) species truncated at the carboxy-terminal (C-terminal) accumulate in pathological inclusions and promote α-syn aggregation and toxicity. Haemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells' vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. In this study, α and ß-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. Long-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, mild motor impairments, and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous α-syn C-terminal truncated species. Similar α-syn fragments were found in vitro in DA iMN9D cells over-expressing α and ß- globins when treated with pre-formed α-syn fibrils. Our study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells' loss in vivo and the formation of C-terminal α-syn fragments.

Maspin overexpression correlates with positive response to primary chemotherapy in ovarian cancer patients.

OBJECTIVE: Maspin is a member of the serine protease inhibitor superfamily. Experimental studies revealed that maspin suppresses tumor growth, angiogenesis, invasion and metastasis. We examined maspin expression in human ovarian tumors and relation between maspin expression and clinicopathological features as well as the role of maspin in predicting clinical outcome in patients with ovarian cancer. METHODS: Tissue samples consisted of 42 benign tumors, 10 borderline (LMP) tumors, 76 ovarian carcinomas, 8 Krukenberg tumors and 32 normal tissues. Immunoblot analysis was performed to evaluate the relative expression of maspin/beta-actin. RESULTS: Relative maspin level was significantly higher in patients with LMP tumors (median 0.74) and early stages ovarian cancers (median 0.46) when compared with healthy tissues (median 0.03), those with benign (median 0.23) and metastatic tumors (median 0.22). Overexpression of maspin was found to correlate with the early stage of the disease (p=0.001), non-serous subtype of ovarian cancer (p=0.03) and positive response to chemotherapy (p=0.02). A statistically significant longer PFS was seen in women with high as compared with low expression of maspin (p=0.03). CONCLUSIONS: Maspin is upregulated in borderline tumors and the early stages of ovarian carcinoma and then significantly downregulated with malignant transformation. High expression may paradoxically promote the invasion and metastasis of ovarian carcinomas. Our study revealed that maspin expression could play an important role in predicting the results of treatment of ovarian cancer patients.

Expression of human HtrA1, HtrA2, HtrA3 and TGF-beta1 genes in primary endometrial cancer.

The HtrA family of serine proteases takes part in cellular stress response including heat shock, inflammation and cancer. Downregulation of human HtrA1 and HtrA3 genes has been reported in some cancers, including endometrial cancer (EC), suggesting a tumor-suppressor role for both genes. The mechanism of the HtrA function is not known, however, evidence exists showing that both HtrA1 and HtrA3 regulate biological processes by modulating TGF-beta signaling. In the presented study the expression of human HtrA1, HtrA2, HtrA3 and TGF-beta1 genes was examined in 124 endometrial tissue specimens including 88 cancers and 36 normal endometria. The expression of the tested genes was evaluated at mRNA and protein levels by semi-quantitative RT-PCR and western blotting methods, respectively. Our results showed significant decrease of HtrA1 and HtrA3 mRNA and protein levels in EC compared to normal tissues. The most dramatic decrease was found for HtrA3 at both mRNA and protein levels (3.2- and 5.6-fold, respectively). Moreover, the HtrA3 protein (short isoform) was not detected in 19% of the cancers, and its level decreased from the premenopausal to the postmenopausal group. The HtrA2 protein levels were significantly lower in EC tissues compared to normal tissues. We also found a significant increase of the TGF-beta1 protein level in EC as well as a significant negative correlation between HtrA1/2/3 and TGF-beta1 relative protein levels. Our results showing downregulation of HtrA1 and HtrA3 gene expression support previous studies suggesting a tumor suppressor role for these genes. Furthermore, our data suggest that HtrA2 may be involved in EC development as well as suggest the involvement of HtrA1, HtrA2 and HtrA3 in the inhibition of TGF-beta signaling in endometrial tissues.

Efficient RT-QuIC seeding activity for α-synuclein in olfactory mucosa samples of patients with Parkinson's disease and multiple system atrophy.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of α-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM). METHODS: We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant α-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce α-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP. RESULTS: Our results showed that a significant percentage of MSA and PD samples induced α-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination. CONCLUSIONS: Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for α-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages.

Changes in expression of serine proteases HtrA1 and HtrA2 during estrogen-induced oxidative stress and nephrocarcinogenesis in male Syrian hamster.

Serine proteases HtrA1 and HtrA2 are involved in cellular stress response and development of several diseases, including cancer. Our aim was to examine the involvement of the HtrA proteins in acute oxidative stress response induced in hamster kidney by estrogen treatment, and in nephrocarcinogenesis caused by prolonged estrogenization of male Syrian hamster. We used semi-quantitative RT-PCR to estimate the HtrA1 and HtrA2 mRNA levels in kidney tissues, and Western blotting to monitor the amount of the HtrA proteins. Within the first five hours following estrogen administration both HtrA1 mRNA and the protein levels were increased significantly. No changes in the expression of HtrA2 were observed. This indicates that HtrA1 may be involved in the response against oxidative stress induced by estrogen treatment in hamster kidney. During prolonged estrogenization, a significant reduction of the HtrA1 mRNA and protein levels was observed after 6 months of estradiol treatment, while the expression of HtrA2 was significantly elevated starting from the third month. This suggests an involvement of the HtrA proteins in estrogen-induced nephrocarcinogenesis in hamster. Using fluorescence in situ hybridization we localized the HtrA1 gene at the qb3-4 region of Syrian hamster chromosome 2, the region known to undergo a nonrandom deletion upon prolonged estrogenization. It is possible that the reduced level of HtrA1 expression is due to this chromosomal aberration. A full-length cDNA sequence of the hamster HtrA1 gene was obtained. It codes for a 50 kDa protein which has 98 and 96% identity with mouse and human counterparts, respectively.

[Characterization of the HtrA family of proteins]. / Charakterystyka bialek z rodziny HtrA.

The HtrA family of proteins consists of evolutionary conserved serine proteases, which are homologues of the HtrA protein from a model bacterium Escherichia coli. They are widely distributed among organisms, prokaryotic as well as eukaryotic including humans. HtrA proteins participate in defense against stresses causing aberrations in protein structure, for example heat or oxidative stress. At least four human homologues have been identified. They are involved in cell growth and differentiation, apoptosis, and disturbances in their function may induce carcinogenesis, arthritic and neurodegenerative disorders. This article summarizes recent studies regarding the HtrA family of proteins, their structure, regulation and function. It also presents practical applications of this knowledge and perspective of its use in the future.

α-Synuclein Amyloids Hijack Prion Protein to Gain Cell Entry, Facilitate Cell-to-Cell Spreading and Block Prion Replication.

The precise molecular mechanism of how misfolded α-synuclein (α-Syn) accumulates and spreads in synucleinopathies is still unknown. Here, we show the role of the cellular prion protein (PrPC) in mediating the uptake and the spread of recombinant α-Syn amyloids. The in vitro data revealed that the presence of PrPC fosters the higher uptake of α-Syn amyloid fibrils, which was also confirmed in vivo in wild type (Prnp +/+) compared to PrP knock-out (Prnp -/-) mice. Additionally, the presence of α-Syn amyloids blocked the replication of scrapie prions (PrPSc) in vitro and ex vivo, indicating a link between the two proteins. Indeed, whilst PrPC is mediating the internalization of α-Syn amyloids, PrPSc is not able to replicate in their presence. This observation has pathological relevance, since several reported case studies show that the accumulation of α-Syn amyloid deposits in Creutzfeldt-Jakob disease patients is accompanied by a longer disease course.

The N-terminal region of HtrA heat shock protease from Escherichia coli is essential for stabilization of HtrA primary structure and maintaining of its oligomeric structure.

HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE, indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide. Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.

Synthetic prions and other human neurodegenerative proteinopathies.

Transmissible spongiform encephalopathies (TSE) are a heterogeneous group of neurodegenerative disorders. The common feature of these diseases is the pathological conversion of the normal cellular prion protein (PrP(C)) into a ß-structure-rich conformer-termed PrP(Sc). The latter can induce a self-perpetuating process leading to amplification and spreading of pathological protein assemblies. Much evidence suggests that PrP(Sc) itself is able to recruit and misfold PrP(C) into the pathological conformation. Recent data have shown that recombinant PrP(C) can be misfolded in vitro and the resulting synthetic conformers are able to induce the conversion of PrP(C) into PrP(Sc)in vivo. In this review we describe the state-of-the-art of the body of literature in this field. In addition, we describe a cell-based assay to test synthetic prions in cells, providing further evidence that synthetic amyloids are able to template conversion of PrP into prion inclusions. Studying prions might help to understand the pathological mechanisms governing other neurodegenerative diseases. Aggregation and deposition of misfolded proteins is a common feature of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and other disorders. Although the proteins implicated in each of these diseases differ, they share a common prion mechanism. Recombinant proteins are able to aggregate in vitro into ß-rich amyloid fibrils, sharing some features of the aggregates found in the brain. Several studies have reported that intracerebral inoculation of synthetic aggregates lead to unique pathology, which spread progressively to distal brain regions and reduced survival time in animals. Here, we review the prion-like features of different proteins involved in neurodegenerative disorders, such as α-synuclein, superoxide dismutase-1, amyloid-ß and tau.

Vitreous amino acid concentrations in patients with glaucoma undergoing vitrectomy.

OBJECTIVE: To measure vitreous concentrations of glutamate and other amino acids in patients with glaucoma undergoing vitrectomy. METHODS: Undiluted vitreous samples were collected from patients undergoing vitrectomy at the University of Iowa (Iowa City) between 1997 and 1998 (n = 69). Vitreous concentrations of 16 amino acids, including glutamate, were determined using high-pressure liquid chromatography. Patients with a history of diabetes mellitus were excluded from the analysis. The study group consisted of those with a history of glaucoma (n = 8), and the control group included those with an epiretinal membrane and/or macular hole with no history of glaucoma (n = 17). Comparison of amino acid concentrations between the 2 groups was performed using a multifactor main effects model that adjusted for the effect of 10 selected covariates. Power analysis was done to determine the level of significant difference in amino acid concentrations. RESULTS: The glaucoma group comprised vitreal specimens from patients with primary open-angle (n = 3) and angle-closure glaucomas that included aqueous misdirection (n = 2), uveitis with secondary angle-closure (n = 2), and Axenfeld Rieger syndrome (n = 1). Indications for vitrectomy in this group included epiretinal membrane, retinal detachment, aqueous misdirection, and uveitis. The control group included specimens from patients with a macular hole (n = 11) and epiretinal membrane (n = 7), with 1 eye having both. Surgical indications in controls were macular hole, retinal detachment, and epiretinal membrane. The mean +/- SD levels of vitreous glutamate, glycine, gamma-aminobutyric acid, and alanine were 6.1 +/- 2.4, 16.3 +/- 7.5, 0.8 +/- 0.3, and 260.5 +/- 101.9 microM, respectively, in glaucoma and 5.2 +/- 2.3, 8.5 +/- 2.5, 0.6 +/- 0.2, and 159.5 +/- 54.9 microM in controls (P >.05 for all). None of the 16 amino acid concentrations measured showed a statistically significant difference between glaucoma and controls (P values between.06 and >.99). A power analysis indicated that a 1.8-fold elevation in the glutamate level was needed to reach significance. MAIN OUTCOME MEASURES: Vitreous amino acid concentrations. CONCLUSIONS: None of the 16 amino acids measured, including glutamate, were significantly elevated in the vitreous of glaucomatous eyes compared with controls. Our results are not consistent with the simple hypothesis of glutamate excitotoxicity in glaucoma. Instead, our findings indicate the dynamic nature of extracellular glutamate, whose concentration is dependent on complex mechanisms not yet fully understood. Further studies are needed to fully elucidate the role of glutamate in the pathogenesis of glaucoma.

Variations in the myocilin gene in patients with open-angle glaucoma.

OBJECTIVE: To determine the prevalence and associated phenotype of myocilin (MYOC) coding sequence variations and a specific promoter polymorphism (MYOC.mt1) in patients with glaucoma and glaucoma suspects. METHODS: A consecutive, unselected series of 779 patients (652 with open-angle glaucoma and 127 glaucoma suspects) were recruited from a university medical center and clinically characterized. The coding sequences of the MYOC gene and the MYOC.mt1 locus in the promoter region were screened for sequence variations. We determined the prevalence of MYOC coding sequence mutations and the MYOC.mt1 promoter polymorphism. We also compared the clinical features of individuals with and without mutations and the MYOC.mt1 promoter polymorphism. RESULTS: Plausible disease-causing sequence variations (DCVs) in the MYOC gene were found in 3.0% of the entire group. Such variations were found in patients with most forms of open-angle glaucoma studied. Patients with primary open-angle glaucoma (POAG) who harbored coding sequence DCVs were clinically similar to patients without them. Patients who harbored the rarer allele of the MYOC.mt1 promoter polymorphism were no different in any measure of disease severity from those who harbored the more common allele. CONCLUSIONS: MYOC DCVs were found in approximately 3% of patients with glaucoma and glaucoma suspects. The 2 alleles of the MYOC.mt1 promoter polymorphism were equally distributed among patients with POAG and healthy control subjects. Patients with POAG who harbored the rarer allele of the MYOC.mt1 promoter polymorphism were no different from those with the more common variant in any measure of disease severity. CLINICAL RELEVANCE: Testing for the MYOC.mt1 promoter polymorphism appears to be of no value in the evaluation of patients with glaucoma.

Escherichia coli small heat shock proteins IbpA/B enhance activity of enzymes sequestered in inclusion bodies.

Escherichia coli small heat shock proteins, IbpA/B, function as molecular chaperones and protect misfolded proteins against irreversible aggregation. IbpA/B are induced during overproduction of recombinant proteins and bind to inclusion bodies in E. coli cells. We investigated the effect of DeltaibpA/B mutation on formation of inclusion bodies and biological activity of enzymes sequestered in the aggregates in E. coli cells. Using three different recombinant proteins: Cro-beta-galactosidase, beta-lactamase and rat rHtrA1 we demonstrated that deletion of the ibpA/B operon did not affect the level of produced inclusion bodies. However, in aggregates containing IbpA/B a higher enzymatic activity was detected than in the IbpA/B-deficient inclusion bodies. These results confirm that IbpA/B protect misfolded proteins from inactivation in vivo.

In vitro aggregation assays for the characterization of α-synuclein prion-like properties.

Aggregation of α-synuclein plays a crucial role in the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson disease (PD), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA). The common feature of these diseases is a pathological deposition of protein aggregates, known as Lewy bodies (LBs) in the central nervous system. The major component of these aggregates is α-synuclein, a natively unfolded protein, which may undergo dramatic structural changes resulting in the formation of ß-sheet rich assemblies. In vitro studies have shown that recombinant α-synuclein protein may polymerize into amyloidogenic fibrils resembling those found in LBs. These aggregates may be uptaken and propagated between cells in a prion-like manner. Here we present the mechanisms and kinetics of α-synuclein aggregation in vitro, as well as crucial factors affecting this process. We also describe how PD-linked α-synuclein mutations and some exogenous factors modulate in vitro aggregation. Furthermore, we present a current knowledge on the mechanisms by which extracellular aggregates may be internalized and propagated between cells, as well as the mechanisms of their toxicity.

Prion protein interaction with soil humic substances: environmental implications.

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders caused by prions. Animal TSE include scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids. Effective management of scrapie in many parts of the world, and of CWD in North American deer population is complicated by the persistence of prions in the environment. After shedding from diseased animals, prions persist in soil, withstanding biotic and abiotic degradation. As soil is a complex, multi-component system of both mineral and organic components, it is important to understand which soil compounds may interact with prions and thus contribute to disease transmission. Several studies have investigated the role of different soil minerals in prion adsorption and infectivity; we focused our attention on the interaction of soil organic components, the humic substances (HS), with recombinant prion protein (recPrP) material. We evaluated the kinetics of recPrP adsorption, providing a structural and biochemical characterization of chemical adducts using different experimental approaches. Here we show that HS act as potent anti-prion agents in prion infected neuronal cells and in the amyloid seeding assays: HS adsorb both recPrP and prions, thus sequestering them from the prion replication process. We interpreted our findings as highly relevant from an environmental point of view, as the adsorption of prions in HS may affect their availability and consequently hinder the environmental transmission of prion diseases in ruminants.

Changes in expression of human serine protease HtrA1, HtrA2 and HtrA3 genes in benign and malignant thyroid tumors.

Human HtrA proteins are serine proteases involved in essential physiological processes. HtrA1 and HtrA3 function as tumor suppressors and inhibitors of the TGF-ß signaling pathway. HtrA2 regulates mitochondrial homeostasis and plays a pivotal role in the induction of apoptosis. The aim of the study was to determine whether the HtrA proteins are involved in thyroid carcinogenesis. We used the immunoblotting technique to estimate protein levels of HtrA1, HtrA2, long and short variants of HtrA3 (HtrA3-L and HtrA3-S) and TGF-ß1 in tissues of benign and malignant thyroid lesions, and control groups. We found that the levels of HtrA2 and HtrA3-S were higher in thyroid malignant tumors compared to normal tissues and benign tumors. The HtrA3-L level was increased in malignant tumor tissues compared to benign tumor tissues and control tissues from patients with benign lesions, and elevated in normal tissues from patients with thyroid carcinoma compared to normal tissues from patients with benign lesions. We also compared levels of HtrA proteins in follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC) and found that these types of carcinoma differed in the expression of HtrA3-S and HtrA1. These results indicate the implication of HtrA proteins in thyroid carcinogenesis suggest that HtrA3 variants may play different roles in cancer development, and that the increased HtrA3-L levels in thyroid tissue could be correlated with the development of malignant lesions. The TGF-ß1 levels in tumor tissues were not significantly altered compared to control tissues.

The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures.

The HtrA (DegP) protein from Escherichia coli is a periplasmic protease whose function is to protect cells from the deleterious effects of various stress conditions. At temperatures below 28 degrees C the proteolytic activity of HtrA was regarded as negligible and it was believed that the protein mainly plays the role of a chaperone. In the present work we provide evidence that HtrA can in fact act as a protease at low temperatures. Under folding stress, caused by disturbances in the disulfide bond formation, the lack of proteolytic activity of HtrA lowered the survival rates of mutant strains deprived of a functional DsbA/DsbB oxidoreductase system. HtrA degraded efficiently the unfolded, reduced alkaline phosphatase at 20 degrees C, both in vivo and in vitro. The cleavage was most efficient in the case of HtrA deprived of its internal S-S bond; therefore we expect that the reduction of HtrA may play a regulatory role in proteolysis.

Changes in mRNA and protein levels of human HtrA1, HtrA2 and HtrA3 in ovarian cancer.

OBJECTIVES: Expression of human HtrA1, HtrA2, HtrA3 and TGF-beta1 genes was examined in ovarian tissue specimens including 19 normal ovaries, 20 benign tumors, 7 borderline tumors, 44 cancers and 8 Krukenberg tumors. DESIGN AND METHODS: mRNA and protein levels were evaluated by semi-quantitative RT-PCR and Western-blotting methods, respectively. RESULTS: A statistically significant decrease of HtrA1 and HtrA3 expression in ovarian tumors comparing to normal tissues was observed. A dramatic decrease of HtrA3 mRNA and protein levels in all tumor tissue groups, and a loss of HtrA3 protein in 30% malignant tumors were found. A significant decrease of HtrA1 mRNA, and of HtrA3 mRNA and protein in malignant tumors compared to benign tumors was revealed. HtrA2 expression in tumor tissues was slightly decreased. Expression of TGF-beta1 in tumor tissues was not significantly different compared to control tissues. CONCLUSIONS: Our results show downregulation of HtrA1 and HtrA3 genes' expression in different types of ovarian tumors and give additional evidence that these genes may function as tumor suppressors.