97 research outputs found
Unveiling the folding mechanism of the bromodomains
Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2). Moreover, we report a description of their kinetic folding mechanism, as obtained by careful analysis of stopped-flow and temperature-jump data. The presence of a high energy intermediate for both proteins, suggested by the non-linear dependence of the folding rate on denaturant concentration in the millisec time regime, has been experimentally observed by temperature-jump experiments. Quantitative global analysis of all the rate constants obtained over a wide range of urea concentrations, allowed us to propose a common, three-state, folding mechanism for these two BRDs. Interestingly, the intermediate of BRD4(1) appears to be more stable and structurally native-like than that populated by BRD2(2). Our results underscore the role played by structural topology and sequence in determining and tuning the folding mechanism
Effect of bet missense mutations on bromodomain function, inhibitor binding and stability
Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin
structure. Acetylated lysine residues are specifically recognized by bromodomains,
small protein interaction modules that read these modification in a sequence and acetylation
dependent way regulating the recruitment of transcriptional regulators and chromatin
remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains
are highly druggable protein interaction domains resulting in the development of a
large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of
attention in the oncology field resulting in the rapid translation of early BET bromodomain
inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism
or found in cancer on BET bromodomain function and stability and the influence
of these mutants on inhibitor binding. We found that most BET missense mutations localize
to peripheral residues in the two terminal helices. Crystal structures showed that the three
dimensional structure is not compromised by these mutations but mutations located in
close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding.
Most mutations affect significantly protein stability and tertiary structure in solution, suggesting
new interactions and an alternative network of protein-protein interconnection as a consequence
of single amino acid substitution. To our knowledge this is the first report studying
the effect of mutations on bromodomain function and inhibitor binding
Characterization of human frataxin missense variants in cancer tissues
Human frataxin is an iron binding protein involved in the mitochondrial Fe-S clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumour initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe-S clusters synthesis and utilization. In this study we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions, however some of the variants show a decreased stability and a decreased functional activity in comparison to that of the wild type protein. This article is protected by copyright. All rights reserved
Oligomerization of Sulfolobus solfataricus
The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60–95 °C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques
Structural Stability of Human Protein Tyrosine Phosphatase ρ Catalytic Domain: Effect of Point Mutations
Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ∼4.0 M urea
Chaperones, chaperonins and heat-shock proteins
The protein folding of a nascent polypeptide is
the decoding of the linear information contained
in the primary sequence into the native and functionally
active three-dimensional conformation.
Chaperone proteins and folding catalysts may
contribute to successful folding into the native
and active protein conformation in the crowded
cellular environment, thus avoiding aggregation
of non-native protein forms. Molecular chaperones
in vivo play a pivotal role in the maintenance of
the proteome quality control and in the correct
balance between protein folding and degradation.
The unbalance of the equilibrium between protein
synthesis, protein folding and protein degradation
may contribute to protein misfolding and
aggregation which may lead to the onset of several
degenerative diseases associated with protein
aggregation, such as Alzheimer’s and Huntington’s
disease
Fondamenti di biochimica umana
In quest’opera gli aspetti più importanti della biochimica umana sono stati raggruppati in tre temi unificanti: biomolecole funzionali e strutturali (vitamine,
modulatori dello stress ossidativo, ormoni e lipidi bioattivi, neurotrasmettitori e componenti del citoscheletro); biochimica d’organo (cuore, fegato, apparato
digerente, reni, sangue, apparato riproduttivo, apparato
muscolare, osso, tessuto adiposo, cute e sistema nervoso); biochimica speciale (biochimica del tumore, fecondazione e sviluppo embrionale, metalli, stato redox
e infiammazione, biochimica dei sensi, biochimica della
nutrizione e biochimica clinica
Beta-Sheet-breaker peptides containing alfa, beta-dehydrophenylalanine: synthesis and in vitro activity studies
The synthesis and fibrillogenesis-inhibiting activity of the new peptide derivatives 1–6, containing α,β-unsaturated phenylalanines, are reported. These compounds are related to the pentapeptide Ac-LPFFD-NH2 (iAβ5p), which was designed by Soto and co-workers and is commonly accepted as a lead compound for fibrillogenesis inhibition . Their activities are determined by Thioflavin T binding assay, far-UV circular dichroism (CD) spectroscopy , and SEM; in addition, their structures in solution are studied through far-UV CD and FTIR spectroscopy. The presence of two α,β-unsaturated phenylalanines increases the fibrillogenesis inhibiting activity significantly in comparison with the lead compound. The interactions between the Aβ1–40 and the inhibitors using electrospray ionization mass spectrometry are also studied. The analyses prove the presence of noncovalent complexes of Aβ1–40 with iAβ5p and its derivatives 1–3 with stoichiometries of 1:1 and 2:1, and the results are independent of time and Aβ1–40/inhibitor rati
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