Papers by Joanna Skórko-glonek
Current Pharmaceutical Design, 2012
The HtrA proteases degrade damaged proteins and thus control the quality of proteins and protect ... more The HtrA proteases degrade damaged proteins and thus control the quality of proteins and protect cells against the consequences of various stresses; they also recognize specific protein substrates and in this way participate in regulation of many pathways. In many pathogenic bacteria strains lacking the HtrA function lose virulence or their virulence is decreased. This is due to an increased vulnerability of bacteria to stresses or to a decrease in secretion of virulence factors. In some cases HtrA is secreted outside the cell, where it promotes the pathogen's invasiveness. Thus, the HtrA proteases of bacterial pathogens are attractive targets for new therapeutic approaches aimed at inhibiting their proteolytic activity. The exported HtrAs are considered as especially promising targets for chemical inhibitors. In this review, we characterize the model prokaryotic HtrAs and HtrAs of pathogenic bacteria, focusing on their role in virulence. In humans HtrA1, HtrA2(Omi) and HtrA3 are best characterized. We describe their role in promoting cell death in stress conditions and present evidence indicating that HtrA1 and HtrA2 function as tumor suppressors, while HtrA2 stimulates cancer cell death induced by chemotherapeutic agents. We characterize the HtrA2 involvement in pathogenesis of Parkinson's and Alzheimer's diseases, and briefly describe the involvement of human HtrAs in other diseases. We hypothesize that stimulation of the HtrA's proteolytic activity might be beneficial in therapies of cancer and neurodegenerative disorders, and discuss the possibilities of modulating HtrA proteolytic activity considering the present knowledge about their structure and regulation.
Frontiers in Microbiology, 2019
International Journal of Molecular Sciences
In the Gram-negative bacteria, many important virulence factors reach their destination via two-s... more In the Gram-negative bacteria, many important virulence factors reach their destination via two-step export systems, and they must traverse the periplasmic space before reaching the outer membrane. Since these proteins must be maintained in a structure competent for transport into or across the membrane, they frequently require the assistance of chaperones. Based on the results obtained for the model bacterium Escherichia coli and related species, it is assumed that in the biogenesis of the outer membrane proteins and the periplasmic transit of secretory proteins, the SurA peptidyl–prolyl isomerase/chaperone plays a leading role, while the Skp chaperone is rather of secondary importance. However, detailed studies carried out on several other Gram-negative pathogens indicate that the importance of individual chaperones in the folding and transport processes depends on the properties of client proteins and is species-specific. Taking into account the importance of SurA functions in ba...
Role of the HtrA protease/chaperone in stress tolerance of <em>Campylobacter jejuni</em>
Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some ... more Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaKMm) and the DnaK from the bacterium Escherichia coli (DnaKEc) were functionally similar when assayed in vitro but DnaKMm failed to substitute for DnaKEc in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaKMm and DnaKEc. DnaKMm showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaKEc. Furthermore, it was unable to negatively regulate the E. coli σ32 transcrip-tion factor level under heat shock conditions and poorly bound purified σ32, which is a native substrate of DnaKEc. These observations taken together indicate differences in substrate specific-ity of archaeal and bacterial DnaKs. Structural modeling of DnaKMm showed some structural dif-ferences in the substrate-binding domains of DnaKMm and DnaKEc...
Second Polish-Korean Conference on Protein Folding
Acta Biochimica Polonica, Sep 17, 2007
Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some ... more Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK Mm) and the DnaK from the bacterium Escherichia coli (DnaK Ec) were functionally similar when assayed in vitro but DnaK Mm failed to substitute for DnaK Ec in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK Mm and DnaK Ec. DnaK Mm showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK Ec. Furthermore, it was unable to negatively regulate the E. coli σ 32 transcription factor level under heat shock conditions and poorly bound purified σ 32 , which is a native substrate of DnaK Ec. These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK Mm showed some structural differences in the substrate-binding domains of DnaK Mm and DnaK Ec , which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK Mm was found preferably in high molecular mass oligomeric forms, contrary to DnaK Ec. Oligomers of DnaK Mm could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK Mm .
The Extracellular Bacterial HtrA Proteins as Potential Therapeutic Targets and Vaccine Candidates
Current Medicinal Chemistry, 2017
BACKGROUND An increasing resistance of bacteria to the commonly used antimicrobials forces to sea... more BACKGROUND An increasing resistance of bacteria to the commonly used antimicrobials forces to search for alternative or supportive ways to cure infections. Targeting virulence factors is one of such approaches. The bacterial HtrA proteins are strongly involved in virulence and the lack of functional HtrA in many cases impairs invasiveness of pathogens. HtrAs act by protecting the cells under stressful conditions as well as they take direct part in invasion of the host. The latter function is played predominantly by the recently identified extracellular fraction of HtrA. This review aims to evaluate HtrAs as therapeutic targets, including design of chemical inhibitors and vaccines. METHODS We undertook a thorough search of bibliographic databases for peer-reviewed scientific literature. RESULTS One hundred and sixty-four papers were included in the review. First, we briefly summarized key structural and functional properties of known HtrA proteins with the special focus on the extracellular HtrA fraction. Then we provided an overview of efforts and advancements to target HtrAs of pathogenic bacteria as a promising antimicrobial therapy. In some cases, encouraging results were obtained and application of HtrAspecific inhibitors protected tissues from damage and killed bacteria. Also promising reports concerning the use of HtrA as a protective antigen in several disease models have recently been published. CONCLUSION The findings of this review suggest that the exported HtrA proteins are very attractive therapeutic targets due to their accessibility, significance in virulence and immunogenicity. However, further extensive studies are still needed to develop a safe antimicrobial treatment.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2016
HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation o... more HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limits access both to the PDZ peptide (carboxylate) binding site and to the PD catalytic center. The molecular mechanism of activation is not well understood. To further the knowledge of HtrA2 thermal activation we monitored the dynamics of the PDZ-PD interactions during temperature increase using tryptophan-induced quenching (TrIQ) method. The TrIQ results suggested that during activation the PDZ domain changed its position versus PD inside a subunit, including a prominent change affecting the L3 regulatory loop of PD, and also changed its interactions with the PD of the adjacent subunit (PD*), specifically with its L1* regulatory loop containing the active site serine. The α5 helix of PDZ was involved in both, the intra-and intersubunit changes of interactions and thus seems to play an important role in HtrA2 activation. The amino acid substitutions designed to decrease the PDZ interactions with the PD or PD* promoted protease activity at a wide range of temperatures, which supports the conclusions based on the TrIQ analysis. The model presented in this work describes PDZ movement in relation to PD and PD*, resulting in an increased access to the peptide binding and active sites, and conformational changes of the L3 and L1* loops.
the bacterium Escherichia coli have different substrate specificities
Cellular Microbiology
The HtrA family of chaperones and serine proteases is important for regulating stress responses a... more The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram-negative and Gram-positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell-to-cell junction factors including E-cadherin, occludin, and claudin-8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development.
Mast cells in mastocytosis and allergy – Important player in metabolic and immunological homeostasis
Advances in Medical Sciences
Acta Biochimica Polonica
Mast cells play important role in both innate and adaptive immunity but clonal proliferation of a... more Mast cells play important role in both innate and adaptive immunity but clonal proliferation of abnormal mast cells in various organs leads to mastocytosis. The skin variant of the disease, cutaneous mastocytosis (CM) is the most frequent form of mastocytosis in children. The HtrA proteases are modulators of important cellular processes, including cell signaling and apoptosis, and are connected with development of many pathologies. The above and the observation that mast cells constitutively release the HtrA1 protein, prompted us to investigate a possible involvement of the HtrA proteins in pediatric CM.We assayed the levels of the serum autoantibodies (IgG) against the recombinant HtrA proteins (HtrA1-4) in children with CM (n= 36) and in healthy controls (n= 62). The anti-HtrA IgGs were detected using enzyme linked immunosorbent assay (ELISA) and Western-blotting. In the CM sera the levels of the anti-HtrA1 and anti-HtrA3 autoantibodies were significantly increased compared to the...
Frontiers in microbiology, 2018
The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the... more The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the virulence of the gastric pathogen . Secreted HtrA (HtrA ) cleaves various junctional proteins such as E-cadherin disrupting the epithelial barrier, which is crucial for bacterial transmigration across the polarized epithelium. Recent studies indicated the presence of two characteristic HtrA forms of 55 and 52 kDa (termed p55 and p52, respectively), in worldwide strains. In addition, p55 and p52 were produced by recombinant HtrA , indicating auto-cleavage. However, the cleavage sites and their functional importance are yet unclear. Here, we determined the amino-terminal ends of p55 and p52 by Edman sequencing. Two proteolytic cleavage sites were identified (H46/D47 and K50/D51). Remarkably, the cleavage site sequences are conserved in HtrA from worldwide isolates, but not in other Gram-negative pathogens, suggesting a highly specific assignment in . We analyzed the role of the amino-term...
The role of the LB structural loop and its interactions with the PDZ domain of the human HtrA3 protease
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Journal of Applied Phycology, 2015
Although the appearance of coloured chlorophyll degradation products of higher plants is well kno... more Although the appearance of coloured chlorophyll degradation products of higher plants is well known, knowledge about such compounds produced and released particularly by planktonic algae is still limited. Colourless conditioned media (CM) obtained from autotrophic cultures of unicellular green alga Desmosdemus subspicatus turn red after acidification. The accumulation of red pigments in the medium and the growth rate of algae were inversely correlated. The red, crude solution isolated from CM by dialysis and ion exchange chromatography, and next purified by means of highperformance liquid chromatography, appeared to be a mixture of three compounds with characteristic UV/VIS absorption maxima near 330 and 505 nm. Electrospray ionization (ESI) mass spectrometry analysis revealed that the molecular mass of the most polar and most abundant compound was 637 Da and molecular masses of two other ones were 641 and 607 Da. Addition of 15 N isotope to the culture medium and subsequent mass spectrometry measurements revealed the occurrence of four nitrogen atoms per each molecule. The data suggest that red pigments isolated from algal-conditioned media are chlorophyll degradation compounds, the production of which depends on light intensity, and are released mainly during the stationary phase of growth.
[The extracytoplasmic protein quality control in bacterium Escherichia coli; the role of proteases and the folding factors]
Postepy biochemii, 2008
The proper functioning of extracytoplasmic proteins requires their correct folding after transloc... more The proper functioning of extracytoplasmic proteins requires their correct folding after translocation and reaching a proper destination within the cellular envelope. This process is supervised by the folding factors and proteases, which comprise the protein quality control system. The coordinated action of its components maintains the proper functioning of the cell under physiological conditions and enables the survival of bacteria under stress conditions. In the present work we provide the concise characteristics of the protein quality control system within the cellular envelope with the particular emphasize on the role of proteolysis in maintaining the cellular homeostasis.
Acta biochimica Polonica, 2007
Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and ... more Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spe...
Acta biochimica Polonica, 2006
DsbA is the major oxidase responsible for generation of disulfide bonds in proteins of E. coli en... more DsbA is the major oxidase responsible for generation of disulfide bonds in proteins of E. coli envelope. In the present work we provided the first detailed characterization of disulfide exchange between DsbA and its natural substrate, HtrA protease. We demonstrated that HtrA oxidation relies on DsbA, both in vivo and in vitro. We followed the disulfide exchange between these proteins spectrofluorimetrically and found that DsbA oxidizes HtrA with a 1:1 stoichiometry. The calculated second-order apparent rate constant (kapp) of this reaction was 3.3x10(4)+/-0.6x10(4) M-1s-1. This value was significantly higher than the values obtained for nonfunctional disulfide exchanges between DsbA and DsbC or DsbD and it was comparable to the kapp values calculated for in vitro oxidation of certain non-natural DsbA substrates of eukaryotic origin.
The Journal of biological chemistry, Jan 4, 1997
The HtrA (DegP) protein of Escherichia coli is a heat shock serine protease, essential for cell s... more The HtrA (DegP) protein of Escherichia coli is a heat shock serine protease, essential for cell survival only at temperatures above 42 degrees C. It has been shown by genetic experiments that HtrA is an envelope protease, functioning in the periplasmic space. To clarify the cellular localization of HtrA, E. coli cells were fractionated, and HtrA was not detected by the immunoblotting technique in the periplasm or in the fraction of soluble proteins but was found in the inner membrane. The protein could be partially eluted from the total membrane fraction by a high ionic strength solution, whereas solutions affecting protein conformation released HtrA almost completely. These results, taken together with the evidence showing that HtrA functions in the periplasm, indicate that HtrA is a peripheral membrane protein, localized on the periplasmic side of the inner membrane. As the first step toward solving the problem of HtrA-membrane interactions, the structure of HtrA in the presence o...
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Papers by Joanna Skórko-glonek