Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1.

Heterozygous loss-of-function mutation of the calcium sensing-receptor (CaSR), causes familial hypocalciuric hypercalcemia type 1 (FHH1), a typically benign condition characterized by mild hypercalcemia. In contrast, homozygous mutation of this dimer-forming G-protein coupled receptor manifests as the lethal neonatal severe hyperparathyroidism (NSHPT). To investigate the mechanisms by which CaSR mutations lead to these distinct disease states, we engineered wild-type (WT) and an exon 5-deficient disease-causing mutation, and transfected expression constructs into human embryonic kidney (HEK) cells. WT protein was mainly membrane-expressed whereas the mutant CaSR protein (mCaSR) was confined to the cytoplasm. Co-expression of WT CaSR directed mCaSR to the cell membrane. In assays of CaSR function, increases in extracellular [Ca2+] ([Ca2+]o) increased intracellular [Ca2+] ([Ca2+]i) in cells expressing WT CaSR while the response was reduced in cells co-expressing mutant and WT receptor. Untransfected cells or those expressing mCaSR alone, showed minimal, equivalent responses to increased [Ca2+]o. Immunoprecipitation experiments confirmed an association between mutant and wild-type CaSR. The affinity of the WT CaSR for calcium was three times greater than that of the heterodimer. The maximal functional response to [Ca]o was dependent on localization of CaSR to the membrane level and independent of homo- or heterodimerizations. In summary, these results suggest that heterodimerization of WT and mCaSR receptors, rescues the trafficking defect of the mutant receptors and also reduces the affinity of the WT-mutant heterodimer for [Ca]o. In contrast, the homozygous mutants do not produce functional receptors on cell membrane. These data indicate how substantial differences between signaling of hetero- and homodimeric mutants may lead to profound differences in the severity of disease in heterozygous and homozygous carriers of these mutations.

Improvement in Glycemic Control of Type 2 Diabetes After Successful Treatment of Hepatitis C Virus.

OBJECTIVE: Hepatitis C virus (HCV) infection is associated with diabetes and may worsen glycemic control in patients with diabetes. We aimed to investigate whether eradication of HCV infection with direct-acting antiviral (DAA) agents is associated with improved glycemic control in patients with diabetes. RESEARCH DESIGN AND METHODS: We identified 2,435 patients with diabetes who underwent interferon-free and ribavirin-free DAA-based antiviral treatment for HCV in the national Veterans Affairs health care system. Changes in average hemoglobin A1c (HbA1c) level and use of antidiabetic medications 1 year before and after antiviral treatment were compared between patients who achieved sustained virologic response (SVR) and those who did not. RESULTS: Among patients with elevated baseline HbA1c, the drop in HbA1c associated with antiviral treatment was greater in those who achieved SVR (0.98%) than in those who sustained treatment failure (0.65%) (adjusted mean difference 0.34, P = 0.02). Use of antidiabetic medications decreased more in patients who achieved SVR than in those who sustained treatment failure, especially for the use of insulin, which dropped significantly from 41.3% to 38% in patients achieving SVR compared with a slight increase from 49.8% to 51% in those who sustained treatment failure. CONCLUSIONS: DAA-based eradication of HCV is associated with improved glycemic control in patients with diabetes as evidenced by decreased mean HbA1c and decreased insulin use. These endocrine benefits of SVR provide additional justification for considering antiviral treatment in all patients with diabetes.

Local Anesthetic Systemic Toxicity Complicating Thyroid Biopsy.

The overdiagnosis of thyroid malignancies may be contributing to the increased incidence of these cancers with a relatively stable mortality rate. We present the case of a man with known malignancies, who underwent biopsy of a suspicious thyroid nodule. This procedure was complicated by local anesthetic systemic toxicity (LAST). It is important to address goals of diagnostic testing and treatment with patients, particularly if further evaluation is unlikely to change management or outcomes.

Nicotinamide adenine dinucleotide-induced multimerization of the co-repressor CtBP1 relies on a switching tryptophan.

The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD(+) binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp(318)) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD(+) binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp(318) switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.

The PARP inhibitor PJ34 causes a PARP1-independent, p21 dependent mitotic arrest.

Poly(ADP)-ribose polymerase (PARP) inhibitors modify the enzymatic activity of PARP1/2. When certain PARP inhibitors are used either alone or in combination with DNA damage agents they may cause a G2/M mitotic arrest and/or apoptosis in a susceptible genetic context. PARP1 interacts with the cell cycle checkpoint proteins Ataxia Telangectasia Mutated (ATM) and ATM and Rad3-related (ATR) and therefore may influence growth arrest cascades. The PARP inhibitor PJ34 causes a mitotic arrest by an unknown mechanism in certain cell lines, therefore we asked whether PJ34 conditionally activated the checkpoint pathways and which downstream targets were necessary for mitotic arrest. We found that PJ34 produced a concentration dependent G2/M mitotic arrest and differentially affected cell survival in cells with diverse genetic backgrounds. p53 was activated and phosphorylated at Serine15 followed by p21 gene activation through both p53-dependent and -independent pathways. The mitotic arrest was caffeine sensitive and UCN01 insensitive and did not absolutely require p53, ATM or Chk1, while p21 was necessary for maintaining the growth arrest. Significantly, by using stable knockdown cell lines, we found that neither PARP1 nor PARP2 was required for any of these effects produced by PJ34. These results raise questions and cautions for evaluating PARP inhibitor effectiveness, suggesting whether effects should be considered not only on PARP's diverse ADP-ribosylation independent protein interactions but also on homologous proteins that may be producing either overlapping or distinct effect.

A non-isotopic in vitro assay for histone acetylation.

We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH(2)-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.

Acetylation of cAMP-responsive element-binding protein (CREB) by CREB-binding protein enhances CREB-dependent transcription.

The coactivator function of cAMP-responsive element-binding protein (CREB)-binding protein (CBP) is partly caused by its histone acetyltransferase activity. However, it has become increasingly clear that CBP acetylates both histones and non-histone proteins, many of which are transcription factors. Here we investigate the role of CBP acetylase activity in CREB-mediated gene expression. We show that CREB is acetylated within the cell and that in vitro, CREB is acetylated by CBP, but not by another acetylase, p300/CBP-associated factor. The acetylation sites within CREB were mapped to three lysines within the CREB activation domain. Although inhibition of histone deacetylase activity results in an increase of CREB- or CBP-mediated gene expression, mutation of all three putative acetylation sites in the CREB activation domain markedly enhances the ability of CREB to activate a cAMP-responsive element-dependent reporter gene. Furthermore, these CREB lysine mutations do not increase interaction with the CRE or CBP. These data suggest that the transactivation potential of CREB may be modulated through acetylation by CBP. We propose that in addition to its functions as a bridging molecule and histone acetyltransferase, the ability of CBP to acetylate CREB may play a key role in modulating CREB-mediated gene expression.

Acetylation of the adenovirus-transforming protein E1A determines nuclear localization by disrupting association with importin-alpha.

Posttranslational modifications may alter the biochemical functions of a protein by modifying associations with other macromolecules, allosterically altering intrinsic catalytic activities, or determining subcellular localization. The adenovirus-transforming protein E1A is acetylated by its cellular targets, the co-activators CREB-binding protein, p300, and p300/CREB-binding protein-associated factor in vitro and also in vivo at a single lysine residue (Lys(239)) within a multifunctional carboxyl-terminal domain necessary for both nuclear localization and interaction with the transcriptional co-repressor carboxyl-terminal binding protein (CtBP). In contrast to a previous report, we demonstrate that acetylation of Lys(239) does not disrupt CtBP binding and that 12 S E1A-mediated repression of CREB-binding protein-dependent transcription does not require recruitment of CtBP. Instead we find that the cytoplasmic fraction of E1-transformed 293 cells is enriched for acetylated E1A with relative exclusion from the nuclear compartment. Whereas wild type 12 S E1A binds importin-alpha 3, binding affinity was markedly reduced both by single amino acid substitution mutations and acetylation at Lys(239). This is the first demonstration that acetylation may alter nuclear partitioning by direct interference with nuclear import receptor recognition. The finding that the cytoplasmic fraction of E1A is acetylated indicates that E1A may exert its pleiotropic effects on cellular transformation in part by affecting cytoplasmic processes.

A Drosophila homologue of Sir2 modifies position-effect variegation but does not affect life span.

Control of chromosome structure is important in the regulation of gene expression, recombination, DNA repair, and chromosome stability. In a two-hybrid screen for proteins that interact with the Drosophila CREB-binding protein (dCBP), a known histone acetyltransferase and transcriptional coactivator, we identified the Drosophila homolog of a yeast chromatin regulator, Sir2. In yeast, Sir2 silences genes via an intrinsic NAD(+)-dependent histone deacetylase activity. In addition, Sir2 promotes longevity in yeast and in Caenorhabditis elegans. In this report, we characterize the Drosophila Sir2 (dSir2) gene and its product and describe the generation of dSir2 amorphic alleles. We found that dSir2 expression is developmentally regulated and that dSir2 has an intrinsic NAD(+)-dependent histone deacetylase activity. The dSir2 mutants are viable, fertile, and recessive suppressors of position-effect variegation (PEV), indicating that, as in yeast, dSir2 is not an essential function for viability and is a regulator of heterochromatin formation and/or function. However, mutations in dSir2 do not shorten life span as predicted from studies in yeast and worms.