1461 research outputs found
Sort by
Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori
Get PDFfirst_pagesettingsOrder Article Reprints
Open AccessArticle
Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori
by Marija Zora Mišković 1,†,Marta Wojtyś 2,†,Maria Winiewska-Szajewska 2,3ORCID,Beata Wielgus-Kutrowska 2ORCID,Marija Matković 4,Darija Domazet Jurašin 5ORCID,Zoran Štefanić 5ORCID,Agnieszka Bzowska 2,*ORCID andIvana Leščić Ašler 5,*ORCID
1
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia
2
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland
3
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland
4
Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia
5
Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(14), 7613; https://doi.org/10.3390/ijms25147613
Submission received: 30 April 2024 / Revised: 5 July 2024 / Accepted: 8 July 2024 / Published: 11 July 2024
(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)
Downloadkeyboard_arrow_down Browse Figures Versions Notes
Abstract
The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked
A minimal Fanconi Anemia complex in early diverging fungi
Get PDFanconi Anemia (FA) pathway resolves DNA interstrand cross links (ICL). The FA pathway was initially
recognized in vertebrates, but was later confirmed in other animals and speculated in fungi. FA
proteins FANCM, FANCL and FANCJ are present in Saccharomyces cerevisiae but, their mechanism
of interaction to resolve ICL is still unclear. Unlike Dikarya, early diverging fungi (EDF) possess more
traits shared with animals. We traced the evolutionary history of the FA pathway across Opisthokonta.
We scanned complete proteomes for FA-related homologs to establish their taxonomic distribution
and analyzed their phylogenetic trees. We checked transcription profiles of FA genes to test if they
respond to environmental conditions and their genomic localizations for potential co-localization.
We identified fungal homologs of the activation and ID complexes, 5 out of 8 core proteins, all of the
endonucleases, and deubiquitination proteins. All fungi lack FANCC, FANCF and FANCG proteins
responsible for post-replication repair and chromosome stability in animals. The observed taxonomic
distribution can be attributed to a gradual degradation of the FA pathway from EDF to Dikarya. One
of the key differences is that EDF have the ID complex recruiting endonucleases to the site of ICL.
Moreover, 21 out of 32 identified FA genes are upregulated in response to different growth conditions.
Several FA genes are co-localized in fungal genomes which also could facilitate co-expression. Our
results indicate that a minimal FA pathway might still be functional in Mucoromycota with a gradual
loss of components in Dikarya ancestors
Roles of SNORD115 and SNORD116 ncRNA clusters during neuronal differentiation
Get PDFIn the snoRNA host gene SNHG14, 29 consecutive introns each generate SNORD116, and 48 tandem introns encode SNORD115. Loss of SNORD116 expression, but not of SNORD115, is linked to the neurodevelopmental disease Prader-Willi syndrome. SNORD116 and SNORD115 resemble box C/D small nucleolar RNAs (snoRNAs) but lack known targets. Both were strongly accumulated during neuronal differentiation, but with distinct mechanisms: Increased host-gene expression for SNORD115 and apparent stabilization for SNORD116. For functional characterization we created cell lines specifically lacking the expressed, paternally inherited, SNORD115 or SNORD116 cluster. Analyses during neuronal development indicates changes in RNA stability and protein synthesis. These data suggest that the loss of SNORD116 enhances some aspects of developmental timing of neuronal cells. Altered mRNAs include MAGEL2, causal in the PWS-like disorder Schaaf-Yang syndrome. Comparison of SNORD115 and SNORD116 mutants identifies small numbers of altered mRNAs and ncRNAs. These are enriched for functions potentially linked to PWS phenotypes and include protocadherins, which are key cell signalling factors during neurodevelopment
Mice, rats, and guinea pigs differ in FMOs expression and tissue concentration of TMAO, a gut bacteria-derived biomarker of cardiovascular and metabolic diseases
Get PDFIntroduction Increased plasma trimethylamine oxide (TMAO) is observed in cardiovascular and metabolic diseases, originating from the gut microbiota product, trimethylamine (TMA), via flavincontaining monooxygenases (FMOs)-dependent oxidation. Numerous studies have investigated the association between plasma TMAO and various pathologies, yet limited knowledge exists regarding tissue concentrations of TMAO, TMAO precursors, and interspecies variability. Methods Chromatography coupled with mass spectrometry was employed to evaluate tissue concentrations of TMAO and its precursors in adult male mice, rats, and guinea pigs. FMO mRNA andprotein levels were assessed through PCR and Western blot, respectively. Results Plasma TMAOlevelsweresimilar amongthe studied species. However, significant differences in tissue concentrations of TMAO were observed between mice, rats, and guinea pigs. The rat renal medulla exhibited the highest TMAO concentration, while the lowest was found in the mouse liver. Mice demonstrated significantly higher plasma TMA concentrations compared to rats and guinea pigs, with the highest TMA concentration found in the mouserenal medulla and the lowest in the rat lungs. FMO5 exhibited the highest expression in mouseliver, while FMO3 was highly expressed in rats. Guinea pigs displayed low expression of FMOsinthis tissue
Extracellular vesicles of Janthinobacterium lividum as violacein carriers in melanoma cell treatment
Get PDFViolacein is a natural indole-derived purple pigment of microbial origin that has attracted attention for its remarkable biological properties. Due to its poor solubility in aqueous media, most studies of this pigment use
extracts of the compound obtained with common solvents. Violacein is also transported in bacterial extracellular
vesicles (EVs) and transferred via this type of carrier remains stable in an aqueous environment. This paper is the
first to present an in-depth study of Janthinobacterium lividum EVs as violacein carriers. J. lividum EVs were
studied for their contribution to violacein translocation, size, morphology and protein composition. The production
of violacein encapsulated in EVs was more efficient than the intracellular production of this compound. The
average size of the violacein-containing EVs was 124.07±3.74 nm. Liquid chromatography-tandem mass
spectrometry analysis (LC-MS/MS) revealed 932 proteins common to three independent EVs isolations. The high1
proportion of proteins with intracellular localisation, which are involved in many fundamental cellular processes,
suggests that J. lividum EVs could be generated in a cell lysis model, additionally stimulated by violacein
production. Using human keratinocytes and melanoma cell lines, it was confirmed that J. lividum EVs are able to
react with and deliver their cargo to mammalian cells. The EVs-delivered violacein was shown to retain its activity
against melanoma cells, and the dose and timing of treatment can be selected to target only cancer cells. The
characterisation of J. lividum EVs, described in the following paper, represents a milestone for their future potential anti-cancer applicatio
Detection of positive selection acting on protein surfaces at the whole-genome scale in the human malaria parasite Plasmodium falciparum.
Get PDFThe host–parasite evolutionary arms race is a fundamental process with medical implications. During this pro-
cess, the host develops parasite resistance, and the parasite develops host immune evasion strategies. Thus, this
process accelerates relevant protein evolution. This study test hypothesizes that proteins subject to sequence
evolution structural constraints play a crucial role and that these constraints hinder the modification of such
proteins in this process. These hypotheses were tested using Plasmodium falciparum model and evaluated protein
structures predicted for the entire proteome by the AlphaFold method. Based on dN/dS test results and
P. falciparum and P. reichenowi comparisons, the presented approach identified proteins subject to purifying
selection acting on the whole sequence and buried residues (dN < dS) and positive selection on nonburied
residues. Of the 26 proteins, some known antigens (ring-exported protein 3, RAP protein, erythrocyte binding
antigen-140, and protein P47) targeted by the host immune system are promising vaccine candidates. The set
also contained 11 enzymes, including FIKK kinase, which modifies host proteins. This set was compared with
genes for which the dN/dS test suggested that positive selection acts on the whole gene (i.e., dN > dS). The
present study found that such genes encode enzymes and antigenic vaccine candidates less frequently than genes
for which evolution is not subject to selection constraints and positive selection acts on only exposed residues.
The analysis was repeated comparing P. falciparum with P. alderi, which is more distantly related. The study
discusses the potential implications of the presented methodology for rational vaccine design and the parasi-
tology and evolutionary biology fields
Prefoldin 2 contributes to mitochondrial morphology and function
Get PDFPrefoldin is an evolutionarily conserved co-chaperone of the tailless complex polypeptide 1 ring complex (TRiC)/chaperonin containing tailless complex 1 (CCT). The prefoldin complex consists of six subunits that are
known to transfer newly produced cytoskeletal proteins to TRiC/CCT for folding polypeptides. Prefoldin function
was recently linked to the maintenance of protein homeostasis, suggesting a more general function of the co-chaperone during cellular stress conditions. Prefoldin acts in an adenosine triphosphate (ATP)-independent manner, making it a suitable candidate to operate during stress conditions, such as mitochondrial dysfunction. Mitochondrial function depends on the production of mitochondrial proteins in the cytosol. Mechanisms that sustain cytosolic protein
homeostasis are vital for the quality control of proteins destined for the organelle and such mechanisms among others include chaperones.
We analyzed consequences of the loss of prefoldin subunits on the cell proliferation and survival of Saccharomyces cerevisiae upon exposure to various cellular stress conditions. We found that prefoldin subunits support
cell growth under heat stress. Moreover, prefoldin facilitates the growth of cells under respiratory growth conditions.
We showed that mitochondrial morphology and abundance of some respiratory chain complexes was supported
by the prefoldin 2 (Pfd2/Gim4) subunit. We also found that Pfd2 interacts with Tom70, a receptor of mitochondrial precursor proteins that are targeted into mitochondria.
Our fndings link the cytosolic prefoldin complex to mitochondrial function. Loss of the prefoldin
complex subunit Pfd2 results in adaptive cellular responses on the proteome level under physiological conditions
suggesting a continuous need of Pfd2 for maintenance of cellular homeostasis. Within this framework, Pfd2 might
support mitochondrial function directly as part of the cytosolic quality control system of mitochondrial proteins
or indirectly as a component of the protein homeostasis networ
Conjugates of Aminoglycosides with Stapled Peptides as a Way to Target Antibiotic-Resistant Bacteria
Get PDFThe misuse and overuse of antibiotics led to the
development of bacterial resistance to existing aminoglycoside (AMG) antibiotics and limited their use. Consequently, there is a growing need to develop effective antimicrobials against multidrugresistant
bacteria. To target resistant strains, we propose to combine
2-deoxystreptamine AMGs, neomycin (NEO) and amikacin
(AMK), with a membrane-active antimicrobial peptide anoplin
and its hydrocarbon stapled derivative. The AMG−peptide hybrids were conjugated using the click chemistry reaction in solution to obtain a non-cleavable triazole linker and by disulfide bridge formation on the resin to obtain a linker cleavable in the bacterial cytoplasm. Homo-dimers connected via disulfide bridges between the N-terminus thiol analogues of anoplin and hydrocarbon stapled anoplin were also synthesized. These hybrid compounds show a notable increase in antibacterial and bactericidal activity, as
compared to the unconjugated ones or their combinations, against Gram-positive and Gram-negative bacteria, especially for the strains resistant to AMK or NEO. The conjugates and disulfide peptide dimers exhibit low hemolytic activity on sheep red blood erythrocytes
Changes in the microbiome of a human and in the simulator of human intestinal microbial ecosystem (SHIME®) in response to a diet and probiotic supplementation
Get PDFBackground. The Simulator of Human Intestinal Microbial Ecosystem (SHIME®) is used to study the behavior of a human microbiome in response to various factors. The aim of this study was to simultaneously demonstrate changes in the microbiome of a human volunteer and in the SHIME® system in response to a change in a diet and probiotic therapy due to a scarcity of published research with similar design.
Results and conclusions. The probiotic therapy resulted in a decrease in fasting insulin and glucose (by 18 % and 13 %, respectively), while the increased fiber intake in the experimental diet seemed to reduce triglyceride, total and LDL cholesterol levels (by 27 %, 15 % and 14 %, respectively) in the volunteer’s blood. Due to the improvement of the volunteer’s metabolic status, changes in her microbiome were observed. Namely, the improvement of glucose homeostasis led to the occurrence of bacteria of the genus Akkermansia in the feces, while the improvement of lipid homeostasis resulted in an increase in the abundance of bacteria of the genus Bacteroides and Bifidobacterium (approx. two and four times, respectively). While changes in the microbiome diversity in the SHIME® luminal fluid (L-SHIME) and in the faeces microbiome were partially similar, the microbiome of SHIME’s intestinal wall (M-SHIME) behaved differently. The characteristic feature of both L- and M-SHIME was a microbiome that differed in its composition compared to the volunteer’s microbiome at parallel sampling points. The results of this study indicate that adjustments to the standard SHIME® experimental protocol should be made to enable the replication of the human microbiome diversity and its changes in the system
Vector-borne parasites in dogs from Ukraine translocated to Poland following Russian invasion in 2022
Get PDFIntroduction: Since 24 February 2022, the day the Russian aggression against Ukraine began, millions of refugees and thousands of pets crossed the Polish-Ukrainian border. Additionally, an unknown number of shelter and stray dogs and cats were rescued and translocated to Poland by private persons and non-profit organizations. The aim of the present study was to examine rescued dogs and cats for presence of canine vector-borne parasites to determine the role of armed conflict in spreading these parasites