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

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

Proto-oncogene Pim-1: structural stability of the variants observed in tumor tissues

Pim-1 kinase belongs to the family of serine/threonine protein kinases (EC 2.7.11.1) encoded by the pim proto-oncogenes (Saris et al., 1991; Hoover et al., 1991; van der Lugt et al., 1995). Pim-1 kinase, originally identified as a common Proviral insertion site in moloney murine leukemia virus-induced T-cell lymphomas in mice (Cuypers et al., 1984), is involved in several signalling pathways and in the regulation of cell cycle progression and apoptosis. The three Pim family members Pim-1, Pim-2 and Pim-3 identified in humans have been reported as signalling protein kinases playing an important role in tumor biology (Anizon et al., 2010). Pim-1, nearly undetectable in normal tissues, is overexpressed in many haematological malignancies and in the cells of several solid tumor. In several cancer tissues Pim-1 variants have been identified and several databases for patterns of somatic mutation in human cancer genomes report mutations in this oncogene (Yuan et al., 2006; Greenman et al., 2007; Forbes et al., 2008; Akagi et al., 2009). Many of these variants are nonsynonymous single nucleotide polymorphisms (nsSNPs), single nucleotide variations occurring in the coding region and leading to amino acid substitutions (Dixit A et al., 2009). In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the Pim-1 kinase. We expressed and purified as soluble recombinant proteins some of the mutants identified in cancer and in the nsSNPs database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to kinase activity, when compared to the wild- type

Single-nucleotide polymorphism of PPARγ, a protein at the crossroads of physiological and pathological processes

Genome polymorphisms are responsible for phenotypic differences between humans and for individual susceptibility to genetic diseases and therapeutic responses. Non-synonymous single-nucleotide polymorphisms (nsSNPs) lead to protein variants with a change in the amino acid sequence that may affect the structure and/or function of the protein and may be utilized as efficient structural and functional markers of association to complex diseases. This study is focused on nsSNP variants of the ligand binding domain of PPARγ a nuclear receptor in the superfamily of ligand inducible transcription factors that play an important role in regulating lipid metabolism and in several processes ranging from cellular differentiation and development to carcinogenesis. Here we selected nine nsSNPs variants of the PPARγ ligand binding domain, V290M, R357A, R397C, F360L, P467L, Q286P, R288H, E324K, and E460K, expressed in cancer tissues and/or associated with partial lipodystrophy and insulin resistance. The effects of a single amino acid change on the thermodynamic stability of PPARγ, its spectral properties, and molecular dynamics have been investigated. The nsSNPs PPARγ variants show alteration of dynamics and tertiary contacts that impair the correct reciprocal positioning of helices 3 and 12, crucially important for PPARγ functioning

b-Sheet interfering molecules acting against b-amyloid aggregation and fibrillogenesis

b-Sheet aggregates and amyloid fibrils rising from conformational changes of proteins are observed in several pathological human conditions. These structures are organized in b-strands that can reciprocally interact by hydrophobic and p–p interactions. The amyloid aggregates can give rise to pathological conditions through complex biochemical mechanisms whose physico-chemical nature has been understood in recent times. This review focuses on the various classes of natural and synthetic small molecules able to act against b-amyloid fibrillogenesis and toxicity that may represent new pharmacological tools in Alzheimer’s diseases. Some peptides, named ‘b-sheet breaker peptides’, are able to hamper amyloid aggregation and fibrillogenesis by interfering with and destabilizing the non native b-sheet structures. Other natural compounds, like polyphenols or indolic molecules such as melatonin, can interfere with b-amyloid peptide pathogenicity by inhibiting aggregation and counteracting oxidative stress that is a key hallmark in Alzheimer’s disease

Alignment of BET bromodomain mutants.

<p>(A) Secondary structure elements are shown at the top of the sequence alignment. Mutated residues are highlighted in red and studied mutations are listed. The conserved asparagine (N391in BRD3(2) numbering) is highlighted in yellow. The green dots represent the residues involved in binding with inhibitor JQ1 (PDB ID: 3ONI, 3S92, 3MXF). The residues underlined in blue are involved in PFI-1binding (PDB ID: 4E96). (B) Location of the mutations. Shown are the first (left) and second bromodomain of BRD2. The mutated residues are highlighted in ball and stick and the position of Cα atoms are shown as a sphere. The main structural elements are labelled.</p

Far-UV CD spectra of wild type bromodomains and mutants.

<p>Far-UV CD spectra were recorded at 20°C in a 0.1-cm quartz cuvette in 20 mM Tris/HCl, pH 7.5 containing 0.20 M NaCl and 0.40 mM DTT, as described in Materials and Methods. Wild type spectra are shown as black solid lines and mutants are coloured as indicated in the figure.</p

Structure of BET mutants and tertiary structure of mutants in solution.

<p>(A) Superimposition of wild type BRD2(1) shown as a ribbon diagram with the mutants BRD2(1) R100L and D161Y shown as protein worm in green and magenta, respectively. The mutated residues are shown in ball and stick representation and main structural elements are labelled. (B) Details of interactions formed by R100 in the wild type compared to the mutated residue. (C) Detailed view of BRD2(1) wild type and D161Y. (D) Superimposition of BRD2(2) shown as a ribbon diagram and the mutant BRD2(2) Q443H shown as protein worm in blue. (E) Comparison of the near UV CD spectra of wild type BRD2(1) and all generated mutants. (F) Comparison of the near UV CD spectra of wild type BRD4(1) and the mutants BRD4(1) A89V. (G) Comparison of the near UV CD spectra of wild type BRD2(2) and the mutants BRD2(2) R419W and Q443H. (H) Comparison of the near UV CD spectra of wild type BRD3(2) and the mutants BRD3(2) H395R. (I) Comparison of the near UV CD spectra of wild type BRD4(2) and the mutants BRD4(2) A420D. Near-UV CD spectra were recorded at 20°C in a 1.0-cm quartz cuvette in 20 mM Tris/HCl, pH 7.5 containing 0.20 M NaCl and 2.00 mM DTT, as described in Materials and Methods.</p

Melting temperatures and thermodynamic parameters for urea-induced unfolding equilibrium of BRDs wild type and mutants measured by far-UV CD and fluorescence spectroscopy.

<p>Melting temperatures and thermodynamic parameters for urea-induced unfolding equilibrium of BRDs wild type and mutants measured by far-UV CD and fluorescence spectroscopy.</p