A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Retinol-binding protein-deficient mice: biochemical basis for impaired vision

We reported previously that mice lacking plasma retinol-binding protein (RBP) are phenotypically normal except that they display impaired vision at the time of weaning. This visual defect is associated with greatly diminished eyecup levels of retinaldehyde and is reversible if the mutants are maintained for several months on a vitamin A-sufficient diet. Here we provide a biochemical basis for the visual phenotype of RBP-deficient mice. This phenotype does not result from inadequate milk total retinol levels since these are not different for RBP-deficient and wild-type mice. The eye, unlike all other tissues that have been examined, takes up dietary retinol very poorly. Moreover, compared to other tissues, the eye displays a strong preference for retinol uptake when retinol is delivered bound to RBP. The poor uptake of dietary retinol by the eye coupled with its marked ability to take up retinol from RBP, we propose, provides a basis for the impaired vision observed in weanling RBP-deficient mice. Further study of the mutants suggests that the impaired vision is reversible because the eyes of mutant mice slowly acquire sufficient retinol from the low levels of retinol present in their circulation either bound to albumin or present in lipoprotein fractions. Thus, the eye is unlike other tissues in the body in that it shows a very marked preference for acquiring retinol needed to support vision from the retinol-RBP complex and is unable to meet adequately its retinol need through uptake of recently absorbed dietary retinol. This provides an explanation for the impaired vision phenotype of RBP-deficient mice.     

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.     

On the association of common and rare genetic variation influencing body mass index: a combined SNP and CNV analysis

Background

As the architecture of complex traits incorporates a widening spectrum of genetic variation, analyses integrating common and rare variation are needed. Body mass index (BMI) represents a model trait, since common variation shows robust association but accounts for a fraction of the heritability. A combined analysis of single nucleotide polymorphisms (SNP) and copy number variation (CNV) was performed using 1850 European and 498 African-Americans from the Study of Addiction: Genetics and Environment. Genetic risk sum scores (GRSS) were constructed using 32 BMI-validated SNPs and aggregate-risk methods were compared: count versus weighted and proxy versus imputation.

Results

The weighted SNP-GRSS constructed from imputed probabilities of risk alleles performed best and was highly associated with BMI (p = 4.3×10⁻¹⁶) accounting for 3% of the phenotypic variance. In addition to BMI-validated SNPs, common and rare BMI/obesity-associated CNVs were identified from the literature. Of the 84 CNVs previously reported, only 21-kilobase deletions on 16p12.3 showed evidence for association with BMI (p = 0.003, frequency = 16.9%), with two CNVs nominally associated with class II obesity, 1p36.1 duplications (OR = 3.1, p = 0.009, frequency 1.2%) and 5q13.2 deletions (OR = 1.5, p = 0.048, frequency 7.7%). All other CNVs, individually and in aggregate, were not associated with BMI or obesity. The combined model, including covariates, SNP-GRSS, and 16p12.3 deletion accounted for 11.5% of phenotypic variance in BMI (3.2% from genetic effects). Models significantly predicted obesity classification with maximum discriminative ability for morbid-obesity (p = 3.15×10⁻¹⁸).

Conclusion

Results show that incorporating validated effect sizes and allelic probabilities improve prediction algorithms. Although rare-CNVs did not account for significant phenotypic variation, results provide a framework for integrated analyses.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-368) contains supplementary material, which is available to authorized users.     

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.     

Cellular retinol-binding protein type III is needed for retinoid incorporation into milk

The physiologic role(s) of cellular retinol-binding protein (CRBP)-III, an intracellular retinol-binding protein that is expressed solely in heart, muscle, adipose, and mammary tissue, remains to be elucidated. To address this, we have generated and characterized CRBP-III-deficient (CRBP-III(-/-)) mice. Mice that lack CRBP-III were viable and healthy but displayed a marked impairment in retinoid incorporation into milk. Milk obtained from CRBP-III(-/-) dams contains significantly less retinyl ester, especially retinyl palmitate, than milk obtained from wild type dams. We demonstrated that retinol bound to CRBP-III is an excellent substrate for lecithin-retinol acyltransferase, the enzyme responsible for catalyzing retinyl ester formation from retinol. Our data indicated that the diminished milk retinyl ester levels arise from impaired utilization of retinol by lecithin-retinol acyltransferase in CRBP-III(-/-) mice. Interestingly, CRBP-I and CRBP-III each appeared to compensate for the absence of the other, specifically in mammary tissue, adipose tissue, muscle, and heart. For CRBP-III(-/-) mice, CRBP-I protein levels were markedly elevated in adipose tissue and mammary gland. In addition, in CRBP-I(-/-) mice, CRBP-III protein levels were elevated in tissues that normally express CRBP-III but were not elevated in other tissues that do not normally express CRBP-III. Our data suggested that CRBP-I and CRBP-III share some physiologic actions within tissues and that each can compensate for the absence of the other to help maintain normal retinoid homeostasis. However, under conditions of high demand for retinoid, such as those experienced during lactation, this compensation was incomplete.     

Nuclear envelope phosphatase 1-regulatory subunit 1 (formerly TMEM188) is the metazoan Spo7p ortholog and functions in the lipin activation pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.