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Germline mutations in CDKN2A (p16) are commonly found in patients with family history of melanoma or personal history of multiple primary melanomas. The p16 tumor suppressor gene regulates cell cycle progression and senescence through binding of cyclin‐dependent kinases (CDK) and also regulates cellular oxidative stress independently of cell cycle control. We identified a germline missense (c.350T>C, p.Leu117Pro) CDKN2A mutation in a patient who had history of four primary melanomas, numerous nevi, and self‐reported family history of melanoma. This particular CDKN2A mutation has not been previously reported in prior large studies of melanoma kindreds or patients with multiple primary melanomas. Compared with wild‐type p16, the p16L117P mutant largely retained binding capacity for CDK4 and CDK6 but exhibited impaired capacity for repressing cell cycle progression and inducing senescence, while retaining its ability to reduce mitochondrial reactive oxygen species. Structural modeling predicted that the Leu117Pro mutation disrupts a putative adenosine monophosphate (AMP) binding pocket involving residue 117 in the fourth ankyrin domain. Identification of this new likely pathogenic variant extends our understanding of CDKN2A in melanoma susceptibility and implicates AMP as a potential regulator of p16. [ABSTRACT FROM AUTHOR]

Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and is a key enzyme of gluconeogenesis and glyconeogenesis and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrates, and notably Homo sapiens, express two FBPase isoforms. The liver isozyme is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis. The muscle isoform is expressed in all cells, and recent studies have demonstrated that its role goes far beyond the enzymatic function, as it can interact with various nuclear and mitochondrial proteins. Even in its enzymatic function, the muscle enzyme is different from the liver isoform, as it is 100-fold more susceptible to allosteric inhibition by AMP and this effect can be abrogated by complex formation with aldolase. All FBPases are homotetramers composed of two intimate dimers: the upper dimer and the lower dimer. They oscillate between two conformational states: the inactive T form when in complex with AMP, and the active R form. Parenthetically, it is noted that bacterial FBPases behave somewhat differently, and in the absence of allosteric activators exist in a tetramer-dimer equilibrium even at relatively high concentrations. [Hines et al. (2007), J. Biol. Chem. 282, 11696-11704]. The T-to-R transition is correlated with the conformation of the key loop L2, which in the T form becomes `disengaged' and unable to participate in the catalytic mechanism. The T states of both isoforms are very similar, with a small twist of the upper dimer relative to the lower dimer. It is shown that at variance with the well studied R form of the liver enzyme, which is flat, the R form of the muscle enzyme is diametrically different, with a perpendicular orientation of the upper and lower dimers. The crystal structure of the muscle-isozyme R form shows that in this arrangement of the tetramer completely new protein surfaces are exposed that are most likely targets for the interactions with various cellular and enzymatic partners. The cruciform R structure is stabilized by a novel `leucine lock', which prevents the key residue, Asp187, from locking loop L2 in the disengaged conformation. In addition, the crystal structures of muscle FBPase in the T conformation with and without AMP strongly suggest that the T-to-R transition is a discrete jump rather than a shift of an equilibrium smooth transition through multiple intermediate states. Finally, using snapshots from three crystal structures of human muscle FBPase, it is conclusively demonstrated that the AMP-binding event is correlated with a β→α transition at the N-terminus of the protein and with the formation of a new helical structure. [ABSTRACT FROM AUTHOR]

Abstract: Adenosine 5′-monophosphate (AMP) inhibits muscle fructose 1,6-bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild-type muscle enzyme, but it binds the fluorescent AMP analogue, 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP), similarly to the wild-type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP-AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission. [Copyright &y& Elsevier]

We established a reverse-genetic approach to identify peroxisomal proteins involved in peroxisomal fatty acid metabolism of Saccharomyces cerevisiae. Putative peroxisomal peripheral membrane proteins were isolated by successive extraction of purified peroxisomes and purified by HPLC and SDS/PAGE. Six proteins were identified by peptide sequence analysis, including acyl-CoA oxidase and a trifunctional enzyme of the peroxisomal ???-oxidation system as well as peroxisomal malate dehydrogenase 3 and carniline acetyhransferase. In addition two previously unknown putative peroxisomal proteins were identified, an unknown 40-kDa protein and a protein which we named Pcs60p, both major constituent of the isolated protein fraction. Pcs60p is encoded by ORF Z36091 of chromosome II from S. cerevisiae and consists of 543 amino acids with a molecular mass of 60.5 kDa. Biochemical, immnofluorescence microscopy and immunocytochemical data confirmed that Pcs60p is a peroxisomal peripheral membrane protein but the protein is also localized in the peroxisomal matrix. Consistent with the intraperoxisomal localization, the consensus sequence for a peroxisomal targeting signal 1 (PTS1) is present at the extreme C-terminus of Pcs60p. Deletion studies revealed that the peroxisornal localization of the protein depends on the presence of this signal sequence. Expression of Pcs60p is highly inducible by oleic acid, however, the protein is dispensable for growth on oleic acid as single carbon source. Pcs60p belongs to the family of proteins which act via an ATP-dependent covalent binding of AMP to their substrates and shows the highest degree of similarity to the Escherichia coli long chain acyl-CoA synthetase. [ABSTRACT FROM AUTHOR]