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E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival.

KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal beta-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

Cerebral cavernous malformations (CCM) are vascular malformations, mostly located in the central nervous system, which occur in 0.1-0.5% of the population. They are characterized by abnormally enlarged and often leaking capillary cavities without intervening neural parenchyma. Some are clinically silent, whereas others cause seizures, intracerebral haemorrhage or focal neurological deficits. These vascular malformations can arise sporadically or may be inherited as an autosomal dominant condition with incomplete penetrance. At least 45% of families affected with cerebral cavernous malformations harbour a mutation in Krev interaction trapped-1 (Krit1) gene (cerebral cavernous malformation gene-1, CCM1). This gene contains 16 coding exons which encode a 736-amino acid protein containing three ankyrin repeats and a FERM domain. Neither the CCM1 pathogenetic mechanisms nor the function of the Krit1 protein are understood so far, although several hypotheses have been inferred from the predicted consequences of Krit1 mutations as well as from the identification of Krit1 as a binding partner of Rap1A, ICAP1A and microtubules. Here, we report the identification of Krit1B, a novel Krit1 isoform characterized by the alternative splicing of the 15th coding exon. We show that the Krit1B splice isoform is widely expressed in mouse cell lines and tissues, whereas its expression is highly restricted in human. In addition, we developed a real-time PCR strategy to accurately quantify the relative ratio of the two Krit1 alternative transcripts in different tissues, demonstrating a Krit1B/Krit1A ratio up to 20% in mouse thymus, but significantly lower ratios in other tissues. Bioinformatic analysis using exon/gene-prediction, comparative alignment and structure analysis programs supported the existence of Krit1 alternative transcripts lacking the 15th coding exon and showed that the splicing out of this exon occurs outside of potentially important Krit1 structural domains but in a region required for association with Rap1A, suggesting a subtle, yet important effect on the protein function. Our results indicate that maintenance of a proper ratio between Krit1A and Krit1B could be functionally relevant and suggest that the novel Krit1B isoform might expand our understanding of the role of Krit1 in CCM1 pathogenesis.

The clinically useful antitumor anthracyclines daunorubicin and doxorubicin represent the results of an original research line of Farmitalia-Carlo Erba begun about thirty years ago as screening of antitumor activity of metabolites isolated from the cultures of several strains of Streptomyces [1]. As all other anthracyclines these molecules consist of an aglycone moiety and an aminosugar joined through a glycosidic bond at C-7; the two structures differ only at C-14 where daunorubicin bears a methyl group and doxorubicin an hydroxymethyl moiety (Figure 1).

Extended X-ray absorption fine structure (EXAFS) has been used to determine the local structure of cobalt in the complex (NCCH2Co(COOCH3)(CO)3)− Na+ in alcoholic solution using the experimental phases and amplitudes derived from NaCo(CO)4. The first coordination sphere consists of 3 C atoms at 1.83(2) and 2 C atoms at 2.13(2) A, in a presumably trigonal bipyramidal arrangement.

A route to penems is described in which dehalogenative reduction of bromohydrins is carried out after ring opening of the penam nucleus. Reduction on some newly synthesized bromopenems suffeerd from low yields. Better yields and acceptable stereoselectivity were obtained on free-hydroxyethyl azetidinone substrates. Some parameters of the reduction are discussed

(+)4-Demethoxydaunomycinone (2) was obtained in five steps through the selective formation of the trifluoromethanesulphonate (4) and its palladium-catalysed reduction.

5′-Deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase was purified to homogeneity from Propionibacterium shermanii by a simplified procedure. The native enzyme has an apparent Mr of 165,000, similar to the enzyme from other sources but larger than previously reported. It consists of two non-identical subunits, of Mr 79,000 and 67,000 respectively. The smaller subunit is apparently not a proteolytic fragment of the larger one. The final preparation usually contained some inactive mutase, bearing a tenaciously bound cobalamin species. This protein proved to be readily separable from apoenzyme by fast protein liquid chromatography on anion-exchange columns.

A series of new syntheses of carboxy compounds derived from cobaltcatalyzed carbonylation of benzyl chloride and ethyl chloroacetate is shown. The choice of experimental parameters, such as reaction medium, type of base and temperature, plays a fundamental role in the selectivity of the carbonylation of benzyl halides. The conditions to achieve highly selective mono- and dicarbonylations of primary and secondary benzyl halides are discussed. The study of the mechanism of the carbonylation of ethyl chloroacetate has revealed the catalytic role of anionic alkoxycarbonylcobalt complexes. These complexes are generated in situ from the corresponding alkyl complexes by nucleophilic attack of the base on a cobalt-coordinated carbonyl. The alkylcobaltcarbonyl complexes and their conjugated anionic alkoxycarbonyl derivatives are shown to be catalysts for new carbonylation reactions. Benzyltrialkylammonium salts, β-substituted styrene oxides, aromatic and vinyl halides can be carbonylated at atmospheric pressure in the presence of these complexes. Some mechanistic hypotheses for cobalt-catalyzed mono- and dicarbonylations of aromatic halides are also discussed.

An efficient resolution of 2,3-epoxyalcohols 2a-b were obtained both by enzymatic hydrolysis and transesterification in organic solvent: (25,3R) 2b and (2R,35) 2a thus obtained are chiral synthons for the synthesis of (+)- Disparlure 1.

Summary Application of the phase-transfer technique to the cobalt carbonyl-catalyzed carbonylation of secondary benzyl halides gives either monocarbonyl and double carbonyl insertion or coupling of organic halides as the major reaction, depending on the experimental conditions. Alcohols or ethers mainly give salts of carboxylic acids. Use of higher pressures of CO association with a hydrocarbon organic phase, favours coupling rather than carbonylation. A possible reaction mechanism is discussed.

A series of new syntheses of carboxy compounds derived from cobaltcatalyzed carbonylation of benzyl chloride and ethyl chloroacetate is shown. The choice of experimental parameters, such as reaction medium, type of base and temperature, plays a fundamental role in the selectivity of the carbonylation of benzyl halides. The conditions to achieve highly selective mono- and dicarbonylations of primary and secondary benzyl halides are discussed. The study of the mechanism of the carbonylation of ethyl chloroacetate has revealed the catalytic role of anionic alkoxycarbonylcobalt complexes. These complexes are generated in situ from the corresponding alkyl complexes by nucleophilic attack of the base on a cobalt-coordinated carbonyl. The alkylcobaltcarbonyl complexes and their conjugated anionic alkoxycarbonyl derivatives are shown to be catalysts for new carbonylation reactions. Benzyltrialkylammonium salts, β-substituted styrene oxides, aromatic and vinyl halides can be carbonylated at atmospheric pressure in the presence of these complexes. Some mechanistic hypotheses for cobalt-catalyzed mono- and dicarbonylations of aromatic halides are also discussed.

A new synthesis of the valuable penem antibiotics FCE 22101 and FCE 22891 which does not require the use of expensive protecting groups is reported. A mixed carbon-silicon protection of two hydroxy groups was used, followed by a selective enzymatic hydrolysis of the resulting 8-O-protected-2-carboxylic ester (18). Lipase from Chromobacterium viscosum and lipoprotein lipase from Pseudomonas sp. gave excellent results in both free or immobilized form.

A new synthesis of the valuable penem antibiotics FCE 22101 and FCE 22891 which does not require the use of expensive protecting groups is reported. A mixed carbon-silicon protection of two hydroxy groups was used, followed by a selective enzymatic hydrolysis of the resulting 8-O-protected-2-carboxylic ester (18). Lipase from Chromobacterium viscosum and lipoprotein lipase from Pseudomonas sp. gave excellent results in both free or immobilized form.