1,720,971 research outputs found
FUNCTIONAL PROTEOMIC ANALYSIS OF COMPLEMENT PROTEINS FROM PATIENTS WITH SYSTEMIC SCLEROSIS
No full textLa sclerodermia (SSc) è una malattia del connettivo che si caratterizza per un eccesivo deposito di collagene a livello di cute e organi interni. Essa è associata a un’anomala risposta immune e a un danno del microcircolo, che è considerato una delle manifestazioni più precoci della malattia.
Il presente progetto di ricerca è nato come il proseguimento di un’analisi proteomica comparativa condotta su siero di soggetti con SSc, che aveva evidenziato il Fattore H (FH) del complemento come una delle proteine differentemente espresse tra pazienti e controlli sani. Dato l’importante ruolo svolto dal FH nella regolazione del complemento sia nella fase fluida sia a livello delle membrane endoteliali dell’ospite, si è deciso di approfondire l’analisi di tale una molecola, per verificare se eventuali anomalie nel FH potessero essere coinvolte nella patogenesi di questa malattia.
Tramite due tecniche complementari, western blot ed ELISA, sono stati confermati gli aumentati livelli di FH nel siero dei malati rispetti ai soggetti sani di controllo. E’ stata inoltre esaminata l’attivazione del complemento nella SSc, tuttora oggetto di dibattito, sia in toto che nella via alternativa, ma non sono state rilevate anomalie significative.
Abbiamo quindi valutato la funzione del FH nella fase fluida e la sua interazione con alcuni ligandi, C3b ed eparina, documentando anche in questo caso l’assenza di anomalie.
Saggi di emolisi hanno invece evidenziato un’aumentata lisi di eritrociti di pecora in seguito all’aggiunta del siero dei malati rispetto ai valori osservati con sieri controllo. Inoltre, abbiamo verificato che la pre-incubazione degli eritrociti con FH, commerciale o purificato da soggetti sani, ne preveniva la lisi quando questi venivano incubati con il siero dei pazienti.
La ricerca di anticorpi diretti contro il FH, che è ancora in fase iniziale e pertanto richiede ulteriori approfondimenti e verifiche, ha evidenziato la presenza di autoanticorpi in una piccola percentuale di malati.
Dal momento che l’analisi genetica non ha evidenziato mutazioni a carico del FH, riteniamo che le anomalie sopra riportate potrebbero essere dovute o alla presenza di autoanticorpi, i cui epitopi sono ancora da localizzare, o a modifiche post-traduzionali del FH che potrebbero averne alterato la funzionalità. Anche alterazioni nella dimerizzazione o complessi tra FH e qualcuno dei suoi molteplici ligandi potrebbero avere un ruolo nell’eziopatogenesi della malattia. Infine, i pazienti con SSc potrebbero avere alterazioni a carico di altre molecole che regolano la cascata del complemento e che in modo diretto o indiretto interagiscono con il FH.
Pertanto, pur esplorando solo alcuni aspetti della complessa della rete di interazioni tra i vari componenti del complemento, il presente studio suggerisce che anomalie nella regolazione del complemento potrebbero avere un ruolo nell’eziopatogenesi della SSc e potrebbe quindi essere il punto di partenza per studi funzionali più mirati.Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive collagen deposition in skin and internal organs. An abnormal immunological response and microvascular damage appear to be early events in the pathophysiology of SSc.
This research project follows a previous proteomic comparative analysis on serum, which had identified Factor H as one of the proteins differently expressed between SSc patients and healthy controls. Since FH has a fundamental role either in complement fluid phase regulation and host cell membrane protection, we focused our attention on this molecule, in the attempt of clarifying its eventual role in SSc pathogenesis.
First of all, we confirmed by two complementary techniques, WB and ELISA, increased FH serum levels in SSc patients. We also analyzed complement activation in our patients, whose involvement in SSc is still matter of debate, without detecting significative abnormalities.
No defects were found either in FH fluid phase activity and interaction with some ligands, i.e. C3b and heparin, evaluated comparing purified FH samples from SSc patients and controls.
We observed an high hemolytic activity of SSc sera towards sheep RBCs compared to control sera, which is reversed by pre-incubation with commercial or healthy purified FH.
Anti-FH antibodies were detected in a small percentage of patients, although a further screening on a wider cohort of subjects and the epitope-mapping are still on course.
Since the genetical analysis did not reveal mutations in FH, we think the reported observations might be explained by anti-FH antibodies or post translational modifications which could block FH membrane activity. Also abnormalities in FH dimerization or complexes between FH and any of its many ligands might have a pathogenetic effect. Finally, SSc patients might present alterations in other complement regulators directly or indirectly connected to FH.
In conclusion, our study proposed a new hypothesis on SSc etiopathogenesis and, although not exploring all the existing connection among the complement players and not reaching definitive conclusions, provided some hints about a possible dysfunction in complement regulation, which could be the starting point of more targeted functional studies
Characterization of C–S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavor applications
No full textVolatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C–S lyase enzymatic activities, is of great significance in industrial applications involving food flavors. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5′-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulfur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate β-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavor-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria, however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavor development. Based on our results, future developments can be expected regarding the flavor-forming potential of Lactobacillus C–S lyase and its use in enhancing food flavors
Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective
Full text linkCa2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca2+-binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca2+ sensors is also presented
Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography
No full textSize exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (R-s), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of R-s by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure.This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the R-s of proteins with some indications that are specific for Ca2+ sensor proteins
Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii
No full textToxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention
Biophysical and biochemical characterization of Arabidopsis thaliana Calmodulin-like protein CML14
No full textCalcium (Ca2+) is one of the most important second messengers in eukaryotes. Ca2+ binding proteins can be subdivided into two categories: “Ca2+ buffers” that modulate Ca2+ ion concentrations in cells, and “Ca2+ sensors” that decode Ca2+ signals in a wide array ofphysiological processes in response to external stimuli. Calmodulin (CaM) is the prototypicalexample of Ca2+ sensor proteins in both animals and plants. In addition to conserved CaM,plants possess a unique family of 50 CaM-like proteins (CMLs). Many of these CMLs still remainuncharacterized and the investigation of their biochemical and biophysical properties willprovide insight into Ca2+ signalling in plants. Herein, a detailed characterization of Arabidopsisthaliana CML14 is reported. CML14 is a protein of 148 amino acids with a theoretical molecularweight of 16,579 Da and 50% amino acid sequence identity with AtCaM2. CML14 is predictedto have one functional Ca2+ binding site despite the presence of three EF-hand motifs(Prosite). We overexpressed CML14 in E. coli and analyzed its biochemical and biophysicalcharacteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation,thermal stability and proteolytic susceptibility upon Ca2+ binding. Isothermal titrationcalorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy identified one Ca2+binding site in CML14 and showed that Ca2+ and Mg2+ compete for the same binding site. TheKd values determined by ITC established that CML14 has higher affinity for Ca2+ than forMg2+. Our data were consistent with the sequence based prediction of one functional calciumbinding site. Differential scanning calorimetry (DSC) showed that Ca2+ and Mg2+ have thesame stabilizing effects on protein folding. Apo-CML14 undergoes two thermal unfoldingtransitions, but in the presence of Ca2+ or Mg2+ only one unfolding event at an intermediatetemperature occurs. Limited proteolysis experiments showed that Ca2+ binding affordsprotection against CML14 digestion by trypsin. Surprisingly, CML14 exhibits very fewconformational changes upon calcium binding, which were evaluated by ANS fluorescence andStokes radius measurements in the apo- and Ca2+ bound-forms. These results suggest thatCML14 does not show the characteristics of a classical Ca2+ sensor protein. To betterunderstand the physiological role of CML14 in plants, in vivo analysis will be performed
Biochemical and biophysical characterization of a plant calmodulin: role of the N- and C-lobes in calcium binding, conformational change, and target interaction.
No full textIn plants, transient elevation of intracellular Ca2+ concentration in response to abiotic stress is responsible for glutamate decarboxylase (GAD) activation via association with calmodulin (CaM), an EF-hand protein consisting of two homologous domains (N and C). An unusual 1:2 binding mode of CaM to CaM-binding domains of GAD has long been known, however the contribution of the two CaM domains in target recognition and activation remains to be clarified. Here, we explored the coupling between physicochemical properties of Arabidopsis CaM1 (AtCaM1) and Arabidopsis GAD1 activation, focusing on each AtCaM1 lobe. We found that the four EF-loops of AtCaM1 differently contribute to the ~20 M apparent affinity for Ca2+ and the C-lobe shows a ~6-fold higher affinity than N-lobe (Kdapp 5.6 M and 32 M for C- and N-lobes, respectively). AtCaM1 responds structurally to Ca2+ in a manner similar to vertebrate CaM based on comparison of Ca2+-induced changes in hydrophobicity exposure, secondary structure, and hydrodynamic behavior. Molecular dynamics simulations of AtCaM1 apo and Ca2+-bound reveal that the latter state is significantly less flexible, although regions of the N-lobe remain quite flexible; this suggests the importance of N-lobe for completing the transition to the extended structure of holo-protein, consistent with data from ANS fluorescence, CD spectroscopy, and SEC analysis. Moreover, enzymatic analysis reveal that mutations in the two lobes affect GAD1 activation in similar ways and only intact AtCaM1 can fully activate GAD1. Taken together, our data provide new insights into the CaM lobes role in interactions between CaM and plant GAD
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Systemic Lupus Erythematosus and Strongyloidiasis: a Multifaceted Connection
No full textWe describe a case of systemic lupus erythematosus complicated by strongyloidiasis. The
parasitic infection appeared with diarrhoea, weight loss and peripheral eosinophilia in association
with recurrence of polyarthritis, probably due to a flare of systemic lupus erythematosus.
The literature about the coexistence of systemic lupus erythematosus and
strongyloidiasis has been reviewed
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