We performed exome sequencing to detect somatic mutations in protein-coding regions in seven melanoma cell lines and donor-matched germline cells. All melanoma samples had high numbers of somatic mutations, which showed the hallmark of UV-induced DNA repair. Such a hallmark was absent in tumor sample-specific mutations in two metastases derived from the same individual. Two melanomas with non-canonical BRAF mutations harbored gain-of-function MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) mutations, resulting in constitutive ERK phosphorylation and higher resistance to MEK inhibitors. Screening a larger cohort of individuals with melanoma revealed the presence of recurring somatic MAP2K1 and MAP2K2 mutations, which occurred at an overall frequency of 8%. Furthermore, missense and nonsense somatic mutations were frequently found in three candidate melanoma genes, FAT4, LRP1B and DSC1.

Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli. The proteins were obtained in a homogeneous state without utilization of urea or detergents. On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55-60 kDa. Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa. At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45-50 and 28-30 kDa. At pH 7.0-7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial a-crystallin. Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein. At pH 6.0, both a-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, a-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase. The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperaturedependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits. Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp. A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins.Keywords: small heat shock proteins; phosphorylation; chaperone activity.Human small heat shock proteins (sHsp) form a large group of proteins, consisting of 10 members with a molecular mass in the range of 17-23 kDa [1]. These proteins are grouped together because all contain an a-crystallin domain, of 80-100 amino acid residues, which is located in the C-terminal part of the protein [2,3]. Some sHsp, such as aB-crystallin and Hsp27, are ubiquitous and expressed in practically all tissues [1,2,4,5], whereas other sHsp (such as HspB7 and HspB9) are expressed only in specific tissues [1,4,5]. sHsp tend to form large oligomers that vary in structure and number of monomers [6,7]. These complexes can be formed by identical or nonidentical subunits. Subunits of a-crystallin, Hsp20, Hsp22, and Hsp27 seem to be involved in the formation of different heterooligomeric complexes [8][9][10][11][12]. Hsp27 and aB-crystallin have been analyzed in detail [2-5,13,14], whereas other members of the large superfamily of sHsp are less well characterized.Hsp20 was described by Kato et al.[8] as a byproduct of purification of human aB-crystallin and Hsp27. Hsp20 is expressed...

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Human αB-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of β7 strand of the α-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wildtype counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the β7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of αB-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the β7 strand participates in the intersubunit interaction of four human small heat shock proteins (αB-crystallin, HspB1, HspB6, HspB8) having different structure of β2 strand of α-crystallin domain and different length and composition of variable N-and C-terminal tails.