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Steinernema kraussei (Steiner, 1923) (Rhabditida: Steinernematidae) – the first record from Poland
Get PDFThis study reports the first record of Steinernema kraussei from Poland. The nematode was isolated from coniferous woodlands in 4 localities in central Poland. Preliminary identification of the species was done based on morphometric measurements. To confirm nematode species of the genus Steinernema the result was supported by the description of the ITS regio
A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity
Get PDFBackground: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible
for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid
disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding
its structure and role in AITD. Homology modeling has shown TPO to be composed of different
structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous
preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence
in TPO.
Methods: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells
lacking the propeptide (TPODpro; amino acid residues 21–108) was characterized and its properties compared to
wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical
studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological
investigations using autoantibodies from AITD patients and other epitope-specific antibodies that
recognize conformational determinants on TPO were evaluated for binding to TPODpro by flow cytometry,
immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics
simulation of TPODpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate
the impact of propeptide removal and the role of glycosylation.
Results: The TPODpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPODpro
was enzymatically active and recognized by patients’ autoantibodies and a panel of epitope-specific antibodies,
confirming structural integrity of the two major conformational determinants recognized by autoantibodies.
Faithful intracellular trafficking and N-glycosylation of TPODpro was also maintained. Molecular modeling and
dynamics simulations were consistent with these observations.
Conclusions: Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of
TPODpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and
importantly is recognized by patients’ autoantibodies, is a key advance for purification of substantial quantities of
homogeneous preparation of TPO for crystallization, structural, and immunological studies
Crucial role of perfringolysin O D1 domain in orchestrating structural transitions leading to membrane-perforating pores: a hydrogen-deuterium exchange study.
Get PDFPerfringolysin O (PFO) is a toxic protein that binds to cholesterol-containing membranes, oligomerizes, and forms a β-barrel transmembrane pore, leading to cell lysis. Previous studies have uncovered the sequence of events in this multistage structural transition to a considerable detail, but the underlying molecular mechanisms are not yet fully understood. By measuring hydrogen-deuterium exchange patterns of peptide bond amide protons monitored by mass spectrometry (MS), we have mapped structural changes in PFO and its variant bearing a point mutation during incorporation to the lipid environment. We have defined all regions that undergo structural changes caused by the interaction with the lipid environment both in wild-type PFO, thus providing new experimental constraints for molecular modeling of the pore formation process, and in a point mutant, W165T, for which the pore formation process is known to be inefficient. We have demonstrated that point mutation W165T causes destabilization of protein solution structure, strongest for domain D1, which interrupts the pathway of structural transitions in other domains necessary for proper oligomerization in the membrane. In PFO, the strongest changes accompanying binding to the membrane focus in D1; the C-terminal part of D4; and strands β1, β4, and β5 of D3. These changes were much weaker for PFO(W165T) lipo where substantial stabilization was observed only in D4 domain. In this study, the application of hydrogen-deuterium exchange analysis monitored by MS provided new insight into conformational changes of PFO associated with the membrane binding, oligomerization, and lytic pore formation
Ada response – a strategy for repair of alkylated DNA in bacteria.
Get PDFAlkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA
Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal
Get PDF4-Hydroxy-2-nonenal (HNE) is a reactive �,�-
unsaturated aldehyde generated during oxidative
stress and subsequent peroxidation of polyunsaturated
fatty acids. Here, Werner protein (WRN) was
identified as a novel target for modification by HNE.
Werner syndrome arises through mutations in the
WRN gene that encodes the RecQ DNA helicase
which is critical for maintaining genomic stability.
This hereditary disease is associated with chromosomal
instability, premature aging and cancer predisposition.
WRN appears to participate in the cellular
response to oxidative stress and cells devoid of WRN
display elevated levels of oxidative DNA damage.
We demonstrated that helicase/ATPase and exonuclease
activities of HNE-modified WRN protein were
inhibited both in vitro and in immunocomplexes purified
from the cell extracts. Sites of HNE adduction
in human WRN were identified at Lys577, Cys727,
His1290, Cys1367, Lys1371 and Lys1389. We applied
in silico modeling of the helicase and RQC domains
of WRN protein with HNE adducted to Lys577 and
Cys727 and provided a potential mechanism of the
observed deregulation of the protein catalytic activities.
In light of the obtained results, we postulate
that HNE adduction to WRN is a post-translational
modification, which may affect WRN conformational
stability and function, contributing to features and
diseases associated with premature senescence
A highly sensitive electrochemical genosensor based on Co-porphyrin-labelled DNA.
Get PDFWe report the use of Co-porphyrins as electrochemical tags for a highly sensitive and selective genosensor. An avian influenza virus-based DNA sequence characteristic of H5N1 was detected at femtomolar levels from competing non-complementary sequences through hybridisation with the labeled DNA
Complement components, proteolysis‑related, and cell communication‑related proteins detected in urine proteomics are associated with IgA nephropathy.
Get PDFINTRODUCTION IgA nephropathy (IgAN) is the most common primary glomerulonephritis. The first symptoms of IgAN are erytrocyturia or hematuria, proteinuria, and decline in renal function, or any combination of the above. One of the promising diagnostic methods is urine proteomics.
OBJECTIVES We studied urine proteomics in patients with IgAN and age- and sex‑matched healthy controls. To minimize the risk of protein degradation, we proposed a new protocol for urine collection and preparation.
PATIENTS AND METHODS A total of 30 patients with IgAN and 30 controls were enrolled into the study. Thirty urine samples of the IgAN group were divided into 3 disease pooled samples (DPS I, II, and III) and 30 urine samples of the control group were divided into 3 control pooled samples (CPS I, II, and III). We used isoelectric focusing/liquid chromatography–mass spectrometry/mass spectrometry (IEF/LC‑MS/MS) to detect all proteins larger than 10 kDa.
RESULTS Using qualitative analysis, we identified 761, 951, and 956 proteins in each of the 3 IEF/LC‑MS/MS experiments. The results were combined, yielding a dataset with 1238 proteins identified by at least 2 peptides. The statistical analysis of the quantitative results revealed 18 proteins that were differently populated in the urine of IgAN patients compared with healthy controls. We found increased urinary concentrations of complement components, coagulation factors, extracellular matrix, intracellular, transmembrane, and other proteins in patients with IgAN. Some of them have never been linked to IgAN before.
CONCLUSIONS We demonstrated that urine proteomics is a promising tool for diagnosing and monitoring patients with IgAN
Biology, systematics, and clinical manifestations of Zygomycota infections
Get PDFFungi cause opportunistic, nosocomial, and community-acquired infections. Among fungal infections (mycoses) zygomycoses are exceptionally severe, with a mortality rate exceeding 50 %. Immunocompromised hosts, transplant recipients, and diabetic patients with uncontrolled keto-acidosis and high iron serum levels are at risk. Zygomycota are capable of infecting hosts immune to other filamentous fungi. The infection often follows a progressive pattern, with angioinvasion and metastases. Moreover, current antifungal therapy frequently has an unfavorable outcome. Zygomycota are resistant to some of the routinely used antifungals, among them azoles (except posaconazole) and echinocandins. The typical treatment consists of surgical debridement of the infected tissues accompanied by amphotericin B administration. The latter has strong nephrotoxic side effects, which make it unsuitable for prophylaxis. Delayed administration of amphotericin and excision of mycelium-containing tissues worsens survival prognoses. More than 30 species of Zygomycota are involved in human infections, among them Mucorales is the most abundant. Prognosis and treatment suggestions differ for each species, which makes fast and reliable diagnosis essential. Serum sample PCR-based identification often gives false-negative results; culture-based identification is time-consuming and not always feasible. With the dawn of Zygomycota sequencing projects significant advancement is expected, as in the case of treatment of Ascomycota infections
Fungal genomes tell a story of ecological adaptations
Get PDFOne genome enables a fungus to have various lifestyles and strategies depending on environmental conditions and in the presence of specific counterparts. The nature of their interactions with other living and abiotic elements is a consequence of their osmotrophism. The ability to degrade complex compounds and especially plant biomass makes them a key component of the global carbon circulation cycle. Since the first fungal genomic sequence was published in 1996 mycology has benefited from the technolgical progress. The available data create an unprecedented opportunity to perform massive comparative studies with complex study design variants targeted at all cellular processes
The impact of synthetic analogs of histidine on copper(II) and nickel(II) coordination properties to an albumin-like peptide. Possible leads towards new metallodrugs.
Get PDFThe purpose of our research was to obtain peptidomimetics possessing Cu(II) and Ni(II) binding properties, which would be useful for biomedical applications. In this context we used potentiometry, UV-VIS and CD spectroscopies to characterize the Cu(II) and Ni(II) binding properties of pentapeptide analogs of the N-terminal sequence of histatin 5. The peptides investigated had a general sequence DSXAK-am (am stands for C-terminal amide), with X including His and its three synthetic analogs, (4-thiazolyl)-L-alanine (1), (2-pyridyl)-L-alanine (2), and (pyrazol-1-yl)-L-alanine (3). The heterocyclic nitrogens present in these analogs were significantly more acidic than that of the His imidazole. We found that DSXAK-am peptides were able to bind Cu(II) and Ni(II) and form 4N complexes in a cooperative fashion, with similar affinities. These results indicate that acidic heterocyclic amino acids provide a viable alternative for histidine in peptidomimetics designed for metal ion binding