The emerging role for Cullin 4 family of E3 ligases in tumorigenesis

As a member of the Cullin-RING ligase family, Cullin-RING ligase 4 (CRL4) has drawn much attention due to its broad regulatory roles under physiological and pathological conditions, especially in neoplastic events. Based on evidence from knockout and transgenic mouse models, human clinical data, and biochemical interactions, we summarize the distinct roles of the CRL4 E3 ligase complexes in tumorigenesis, which appears to be tissue- and context-dependent. Notably, targeting CRL4 has recently emerged as a noval anti-cancer strategy, including thalidomide and its derivatives that bind to the substrate recognition receptor cereblon (CRBN), and anticancer sulfonamides that target DCAF15 to suppress the neoplastic proliferation of multiple myeloma and colorectal cancers, respectively. To this end, PROTACs have been developed as a group of engineered bi-functional chemical glues that induce the ubiquitination-mediated degradation of substrates via recruiting E3 ligases, such as CRL4 (CRBN) and CRL2 (pVHL). We summarize the recent major advances in the CRL4 research field towards understanding its involvement in tumorigenesis and further discuss its clinical implications. The anti-tumor effects using the PROTAC approach to target the degradation of undruggable targets are also highlighted.     

Nemaline myopathy caused by mutations in the muscle alpha-skeletal-actin gene

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder characterized by muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle. Disease-causing mutations have been reported in five genes, each encoding a protein component of the sarcomeric thin filament. Recently, we identified mutations in the muscle alpha-skeletal-actin gene (ACTA1) in a subset of patients with NM. In the present study, we evaluated a new series of 35 patients with NM. We identified five novel missense mutations in ACTA1, which suggested that mutations in muscle alpha-skeletal actin account for the disease in approximately 15% of patients with NM. The mutations appeared de novo and represent new dominant mutations. One proband subsequently had two affected children, a result consistent with autosomal dominant transmission. The seven patients exhibited marked clinical variability, ranging from severe congenital-onset weakness, with death from respiratory failure during the 1st year of life, to a mild childhood-onset myopathy, with survival into adulthood. There was marked variation in both age at onset and clinical severity in the three affected members of one family. Common pathological features included abnormal fiber type differentiation, glycogen accumulation, myofibrillar disruption, and "whorling" of actin thin filaments. The percentage of fibers with rods did not correlate with clinical severity; however, the severe, lethal phenotype was associated with both severe, generalized disorganization of sarcomeric structure and abnormal localization of sarcomeric actin. The marked variability, in clinical phenotype, among patients with different mutations in ACTA1 suggests that both the site of the mutation and the nature of the amino acid change have differential effects on thin-filament formation and protein-protein interactions. The intrafamilial variability suggests that alpha-actin genotype is not the sole determinant of phenotype.     

Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay

The fluorophore, Phen Green SK (PGSK), was assessed for its suitability to be used in an assay for ferrous ion transport into membrane vesicles. The long wavelengths of excitation and emission (506 and 520 nm, respectively) enable PGSK fluorescence to be detected in membranes, such as the chloroplast inner envelope, that contain high levels of carotenoids which absorb light at lower wavelengths. At low concentrations of Fe2+, less than 3 microM, the interaction between PGSK and Fe2+ appears to result in both static and dynamic quenching of the PGSK fluorescence. The characteristics of this quenching were used to develop a calibration curve to determine the concentration of free Fe2+ at these low concentrations. Pronounced quenching of PGSK fluorescence entrapped within chloroplast inner envelope membrane vesicles was observed when Fe2+ was added. The extent of quenching of PGSK fluorescence trapped inside asolectin vesicles on Fe2+ addition was much less. The kinetics of the quenching of PGSK fluorescence by Fe2+ in vesicles was quite different from that for PGSK and Fe2+ in solution. Using the calibration curve developed for interaction of PGSK and low Fe2+ concentrations the initial rates of iron transport could be determined for the chloroplast inner envelope membranes.     

Myodegeneration in EDA-A2 transgenic mice is prevented by XEDAR deficiency

EDA-A1 and EDA-A2 are members of the tumor necrosis factor family of ligands. The products of alternative splicing of the ectodysplasin (EDA) gene, EDA-A1 and EDA-A2 differ by an insertion of two amino acids and bind to distinct receptors. The longer isoform, EDA-A1, binds to EDAR and plays an important role in sweat gland, hair, and tooth development; mutations in EDA, EDAR, or the downstream adaptor EDARADD cause hypohidrotic ectodermal dysplasia. EDA-A2 engages the receptor XEDAR, but its role in the whole organism is less clear. We have generated XEDAR-deficient mice by gene targeting and transgenic mice expressing secreted forms of EDA-A1 or EDA-A2 downstream of the skeletal muscle-specific myosin light-chain 2 or skin-specific keratin 5 promoter. Mice lacking XEDAR were indistinguishable from their wild-type littermates, but EDA-A2 transgenic mice exhibited multifocal myodegeneration. This phenotype was not observed in the absence of XEDAR. Skeletal muscle in EDA-A1 transgenic mice was unaffected, but their sebaceous glands were hypertrophied and hyperplastic, consistent with a role for EDA-A1 in the development of these structures. These data indicate that XEDAR-transduced signals are dispensable for development of ectoderm-derived organs but might play a role in skeletal muscle homeostasis.     

Genome-wide linkage analysis replicates susceptibility locus for fasting plasma triglycerides: NHLBI Family Heart Study

Recent reports implicate chromosomal regions linked to inter-individual variation in plasma triglycerides. We conducted genome-wide scans to replicate these linkages and/or identify other loci influencing plasma triglycerides in the NHLBI Family Heart Study (FHS). Data were obtained for 501 three-generational families. Genotyping was done by the Utah Molecular Genetics Laboratory and NHLBI Mammalian Genotyping Service; markers from both were placed on one genetic map. Analysis was done using multipoint variance components linkage. Fasting plasma triglycerides were log-transformed and age-, sex-, and field center-adjusted; suggestive linkage evidence was found on chromosome 8 (LOD=2.80 at 89 cM, marker D8S1141). Further adjustment for waist girth, BMI, diabetes, hypertension, and lipid-lowering drugs suggested linkage regions on chromosomes 6 (LOD=2.29 at 79 cM, marker D6S295) and 15 (LOD=1.85 at 43 cM, marker D15S659). Since HDL is correlated with triglycerides and because it was linked to this region on chromosome 15 in FHS, we created a composite triglyceride–HDL phenotype. The combined phenotype LOD score was 3.0 at the same marker on chromosome 15. Chromosome 15 likely harbors a susceptibility locus with an influence on triglycerides and HDL. Regions on chromosomes 6 and 8 may also contain loci contributing to inter-individual variation in plasma triglycerides.