The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A long-standing question has focused on how GR and other receptors precisely control gene expression. One difficulty in addressing this is that GR function is influenced by multiple factors including ligand and coactivator levels, chromatin state, and allosteric coupling. Moreover, the receptor recognizes an array of DNA sequences that generate a range of transcriptional activities. Such complexity suggests that any single parameter-DNA binding affinity, for example-is unlikely to be a dominant contributor to function. Indeed, a number of studies have suggested that for GR and other receptors, binding affinity toward different DNA sequences is poorly correlated with transcriptional activity. As a step toward determining the factors most predictive of GR function, we rigorously examined the relationship between in vitro GR-DNA binding energetics and in vivo transcriptional activity. We first demonstrate that previous approaches for assessing affinity-function relationships are problematic due to issues of data transformation and linearization. Thus, the conclusion that binding energetics and transcriptional activity are poorly correlated is premature. Using more appropriate analyses, we find that energetics and activity are in fact highly correlated. Furthermore, this correlation can be quantitatively accounted for using simple binding models. Finally, we show that the strong relationship between energetics and transcriptional activity is recapitulated in multiple promoter contexts, cell lines, and chromatin environments. Thus, despite the complexity of GR function, DNA binding energetics are the primary determinant of sequence-specific transcriptional activity.

Triple-negative breast cancers (TNBCs) are among the most aggressive cancers characterized by a high propensity to invade, metastasize and relapse. We previously reported that the TNBC-specific inhibitor, AMPI-109, significantly impairs the ability of TNBC cells to migrate and invade by reducing levels of the metastasis-promoting phosphatase, PRL-3. Here, we examined the mechanisms by which AMPI-109 and loss of PRL-3 impede cell migration and invasion. AMPI-109 treatment or knock down of PRL-3 expression were associated with deactivation of Src and ERK signaling and concomitant downregulation of RhoA and Rac1/2/3 GTPase protein levels. These cellular changes led to rearranged filamentous actin networks necessary for cell migration and invasion. Conversely, overexpression of PRL-3 promoted TNBC cell invasion by upregulating matrix metalloproteinase 10, which resulted in increased TNBC cell adherence to, and degradation of, the major basement membrane component laminin. Our data demonstrate that PRL-3 engages the focal adhesion pathway in TNBC cells as a key mechanism for promoting TNBC cell migration and invasion. Collectively, these data suggest that blocking PRL-3 activity may be an effective method for reducing the metastatic potential of TNBC cells.

Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoproteinubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired. Among these dehydrogenases are the four chain length-specific flavoprotein dehydrogenases of fatty acid ␤-oxidation. In this investigation, two mutations in the ␣ subunit that have been identified in patients were expressed in Escherichia coli. Of the two mutant alleles, ␣T266M and ␣G116R, the former is the most frequent mutation found in patients with ETF deficiency. The crystal structure of human ETF shows that ␣G116 lies in a hydrophobic pocket, under a contact residue of the ␣/␤ subunit interface, and that the hydroxyl hydrogen of ␣T266 is hydrogen Stable expression of the ␣G116R ETF required coexpression of the chaperonins, GroEL and GroES. ␣G116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts. It is unstable and could not be extensively purified. The ␣T266M ETF was purified and characterized after stabilization to proteolysis in crude extracts. Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein.The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF. The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl-CoA dehydrogenases which reduce the flavin to the semiquinone. However, k cat /K m of ETF-QO in a coupled acylCoA:ubiquinone reductase assay with oxidized ␣T266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the ␣T266M ETF semiquinone catalyzed by ETF-QO. Electron transfer flavoproteins (ETF)1 are heterodimeric, FAD-containing proteins that transfer electrons between primary dehydrogenases and respiratory chains in eukaryotic and prokaryotic cells. In mammalian systems, ETF transfers electrons from nine mitochondrial flavoprotein dehydrogenases to the main respiratory chain via the iron-sulfur flavoprotein, ETF-ubiquinone oxidoreductase (ETF-QO) (1). Porcine ETF is apparently closely related to human ETF (2, 3). Both proteins stabilize an anionic flavin semiquinone upon reduction by the flavoprotein dehydrogenases (4, 5). Reduction of ETF to the hydroquinone oxidation state by the dehydrogenases is very slow and not kinetically significant (4, 6). However, kinetic studies of the ETF-QO-catalyzed reduction of ubiquinone by reduced ETF by Ramsay et al. indicated that ETF hydroquinone, and not the semiquinone, is the reductant of ubiquinone (6). Thi...

The lysosomal catabolism of sulfatide requires arylsulfatase A and a specific sphingolipid activator protein, SAP-1. While most patients with metachromatic leukodystrophy have mutations in the gene for arylsulfatase A, some patients have deficient SAP-1, as determined by immunological techniques. We now describe the molecular findings in a patient who died at 22 years of age with SAP-1 deficiency. The DNA polymerase chain reaction was used to amplify regions of cDNA which were subcloned in M13 phage DNA and sequenced by the dideoxy chain-termination method. The patient was found to have a 33-base-pair insertion between nucleotides 777 and -778 (numbered from the A of the ATG initiation codon). No other changes were found in the coding sequence of the cDNA from this patient. At the site of the insertion some normal people have an additional 9 base pairs, which correspond to the last 9 nucleotides at the 3' end of the insertion. The cDNAs from the second-cousin parents were amplified and sequenced, and in both two alleles were identified, one with the 33-base-pair insertion and one with no insertion. Two brothers were found to have only the normal alleles and a sister was found to have the 33-base-pair insertion and a normal allele. The findings confirm studies performed on leukocyte extracts demonstrating normal antigen levels in the two brothers and a lower level in the sister. The presence of i1 additional amino acids in the coding region of mature SAP-1 in this patient causes significant changes in the hydropathy profile compatible with the previous findings at the protein level.The catabolism of most sphingolipids requires both a specific lysosomal enzyme and a specific, relatively low molecular weight protein we call sphingolipid activator protein (SAP) (1). While at least six SAPs have been identified, the exact number is not known. However, it is now obvious that, while most lysosomal disorders are due to defects in lysosomal enzymes, some very serious and fatal disorders are due to defects in SAP (2-5). These include the AB variant of GM2 gangliosidosis, a variant form of metachromatic leukodystrophy, and a variant form of Gaucher disease. While the patients have clear evidence for decreased catabolism of a specific lipid, the lysosomal enzyme activity measured in vitro is normal and there is evidence for decreased levels of a specific SAP in all cases thus far identified (2,5,6).The cDNA coding for the SAP-1 precursor species, called prosaposin by Morimoto et al. (7), has been cloned and sequenced (8,9). Most recently Nakano et al. (10) published the complete corrected sequence, which is in agreement with our unpublished data, except those authors found a 9-base-pair (bp) insertion between nucleotides 777 and 778 (counting from the A of the ATG initiation codon) in clones from normal people. The SAP-1 precursor protein contains four domains of about 80 amino acids, each with similar structural features, including glycosylation sites and proline and cysteine residues (11-13). The domains are prot...

A 28-month-old black male died with severe complications of mental and motor deterioration, seizures, and aspiration. Autopsy demonstrated moderate liver enlargement, normal spleen and kidneys, small testes, and a grossly normal brain. Further examination showed irregular macrogyrae with evidence of a storage or sclerotic process. Thin layer chromatography of the lipids in formalin-fixed tissue demonstrated elevated levels of ceramide trihexoside and possibly sulfatides in liver and a decrease in the ratio of galactosylceramide to sulfatide in brain. Examination of the gangliosides in formalin-fixed brain indicated a slight increase in the percentage of GM1 ganglioside and a clear elevation in GM2 and GM3 gangliosides. Cultured skin fibroblasts had a normal activity for a large number of lysosomal enzymes including arylsulfatase A and galactocerebrosidase. When the cells were loaded with [14C]sulfatide only about 12% of the sulfatide was metabolized after 3 days. Extracts of the cells were subjected to SDS-PAGE and immunoblotting with antisphingolipid activator protein-1 (SAP-1) rabbit antiserum, and no cross-reacting material was detected confirming the diagnosis of metachromatic leukodystrophy caused by SAP-1 deficiency. This patient was clinically more severe than the other patients described previously with this deficiency. Further studies are underway to define the nature of the mutation in this patient.