1,721,138 research outputs found
Substrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from Mycobacterium tuberculosis
No full textMycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis-Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site
Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197
No full textDuring 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19
Polyglutamine is not all: the functional role of the AXH domain in the ataxin-1 protein.
No full textA family of neurodegenerative diseases is associated with anomalous expansion of a polyglutamine tract in the coding region of the corresponding proteins. The current working hypothesis is that polyglutamine diseases are caused by misfolding and aggregation of the proteins with a process dictated by the polyglutamine tracts, although increasing evidence suggests an involvement of the protein context in modulating these properties. Here, we show that the AXH domain of ataxin-1, the protein involved in spinocerebellar ataxia type-1, is the region responsible for the transcriptional repression activity of ataxin-1 and participates in protein aggregation. In vitro, the isolated domain undergoes a conformational transition towards a beta-enriched structure associated with aggregation and amyloid fibre formation spontaneously and without need for destabilizing conditions. Using a transfected cell line, we demonstrate that, while determined by polyglutamine expansion, ataxin-1 aggregation is noticeably reduced by deletion of AXH or by replacement with the homologous sequence from the transcription factor HBP1, which has no known tendency to aggregate. These results provide the first direct evidence of an involvement of a region other than the polyglutamine tract in polyglutamine pathologies
Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3
No full textExpansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate
Binding of alpha−actinin to titin: implications for Z−disk assembly
No full textTitin is an exceptionally large protein (M.Wt. approximately 3 MDa) that spans half the sarcomere in muscle, from the Z−disk to the M−line. In the Z−disk, it interacts with alpha−actinin homodimers that are a principal component of the Z−filaments linking actin filaments. The interaction between titin and alpha−actinin involves repeating approximately 45 amino acid sequences (Z−repeats) near the N−terminus of titin and the C−lobe of the C−terminal calmodulin−like domain of alpha−actinin. The conformation of Z−repeat 7 (ZR7) of titin when complexed with the 73−amino acid C−terminal portion of alpha−actinin (EF34) was studied by heteronuclear NMR spectroscopy using (15)N−labeling of ZR7 and found to be helical over a stretch of 18 residues. Complex formation resulted in the protection of one site of preferential cleavage of EF34 at Phe14−Leu17, as determined by limited proteolysis experiments coupled to mass spectrometry measurements. Intermolecular NOEs show Val16 of ZR7 to be positioned close in space to the backbone of EF34 around Phe14. These observations suggest that the mode of binding of ZR7 to EF34 is similar to that of troponin I to troponin C and of peptide C20W to calmodulin. These complexes would appear to represent a general alternative binding mode of calmodulin−like domains to target peptide
Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation
No full textInhibition of the thioredoxin system is a basis for the antileukemic potential of 13-hydroxy-15-oxo-zoapatlin
No full textThe mammalian thioredoxin (Trx) system, composed of Trx, Trx reductase (TrxR), and NADPH, is the most
important thiol system involved in the redox control of signaling and regulatory proteins in apoptosis and
cell proliferation. Here we addressed the inhibition of the Trx system by 13-hydroxy-15-oxo-zoapatlin (OZ), a
nor-kaurane diterpene previously shown to possess proapoptotic potential and to cause cell cycle arrest in
leukemia cells. OZ was found, by both biochemical and mass spectrometry-based approaches, to target Trx1
and TrxR in a cell-free system. In particular, the formation of reversible OZ adducts to Trx1 Cys35, Cys62, and
Cys73 was demonstrated. We next showed that OZ efficiently inhibited Trx and TrxR catalytic activity in
Molt4 cells. The occurrence of oxidative modifications of Trx molecules was assessed by “redoxWestern blot”
analyses. OZ-mediated Trx oxidation resulted in apoptosis signaling kinase-1 release and activation of
downstream JNK and p38 pathways. By means of specific inhibitors of these two stress-activated protein
kinases, we demonstrated that the JNK pathway plays a major role in determining the apoptotic fate of OZexposed
cells, whereas p38 activation seems to be involved mainly in OZ-induced G2/M block
Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway
No full textAnnexin A1 (ANXA1) is a Ca2+-binding protein over-expressed in pancreatic cancer (PC). We recently reported that extracellular ANXA1 mediates PC cell motility acting on Formyl Peptide Receptors (FPRs). Here, we describe other mechanisms by which intracellular ANXA1 could mediate PC progression. We obtained ANXA1 Knock-Out (KO) MIA PaCa-2 cells using the CRISPR/Cas9 genome editing technology. LC-MS/MS analysis showed altered expression of several proteins involved in cytoskeletal organization. As a result, ANXA1 KO MIA PaCa-2 partially lost their migratory and invasive capabilities with a mechanism that appeared independent of FPRs. The acquisition of a less aggressive phenotype has been further investigated in vivo. Wild type (WT), PGS (scrambled) and ANXA1 KO MIA PaCa-2 cells were engrafted orthotopically in SCID mice. No differences were found about PC primary mass, conversely liver metastatization appeared particularly reduced in ANXA1 KO MIA PaCa-2 engrafted mice. In summary, we show that intracellular ANXA1 is able to preserve the cytoskeleton integrity and to maintain a malignant phenotype in vitro. The protein has a relevant role in the metastatization process in vivo, as such it appears attractive and suitable as prognostic and therapeutic marker in PC progression
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