Acetylation-Specific Interference by Anti-Histone H3K9ac Intrabody Results in Precise Modulation of Gene Expression.

Among Histone post-translational modifications (PTMs), lysine acetylation plays a pivotal role in the epigenetic regulation of gene expression, mediated by chromatin modifying enzymes. Due to their activity in physiology and pathology, several chemical compounds have been developed to inhibit the function of these proteins. However, the pleiotropy of these classes of proteins represents a weakness of epigenetic drugs. Ideally, a new generation of epigenetic drugs should target with molecular precision individual acetylated lysines on the target protein. We exploit a PTM-directed interference, based on an intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), to test the hypothesis that targeting H3K9ac yields more specific effects than inhibiting the corresponding HAT enzyme that installs that PTM. In yeast scFv-58F modulates, gene expression in a more specific way, compared to two well-established HAT inhibitors. This PTM-specific interference modulated expression of genes involved in ribosome biogenesis and function. In mammalian cells, the scFv-58F induces exclusive changes in the H3K9ac-dependent expression of specific genes. These results suggest the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the writer enzyme to acting on the PTM.

Selection and Modelling of a New Single-Domain Intrabody Against TDP-43.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated to deteriorating motor and cognitive functions, and short survival. The disease is caused by neuronal death which results in progressive muscle wasting and weakness, ultimately leading to lethal respiratory failure. The misbehaviour of a specific protein, TDP-43, which aggregates and becomes toxic in ALS patient's neurons, is supposed to be one of the causes. TDP-43 is a DNA/RNA-binding protein involved in several functions related to nucleic acid metabolism. Sequestration of TDP-43 aggregates is a possible therapeutic strategy that could alleviate or block pathology. Here, we describe the selection and characterization of a new intracellular antibody (intrabody) against TDP-43 from a llama nanobody library. The structure of the selected intrabody was predicted in silico and the model was used to suggest mutations that enabled to improve its expression yield, facilitating its experimental validation. We showed how coupling experimental methodologies with in silico design may allow us to obtain an antibody able to recognize the RNA binding regions of TDP-43. Our findings illustrate a strategy for the mitigation of TDP-43 proteinopathy in ALS and provide a potential new tool for diagnostics.

Protein Structural Information and Evolutionary Landscape by In Vitro Evolution.

Protein structure is tightly intertwined with function according to the laws of evolution. Understanding how structure determines function has been the aim of structural biology for decades. Here, we have wondered instead whether it is possible to exploit the function for which a protein was evolutionary selected to gain information on protein structure and on the landscape explored during the early stages of molecular and natural evolution. To answer to this question, we developed a new methodology, which we named CAMELS (Coupling Analysis by Molecular Evolution Library Sequencing), that is able to obtain the in vitro evolution of a protein from an artificial selection based on function. We were able to observe with CAMELS many features of the TEM-1 beta-lactamase local fold exclusively by generating and sequencing large libraries of mutational variants. We demonstrated that we can, whenever a functional phenotypic selection of a protein is available, sketch the structural and evolutionary landscape of a protein without utilizing purified proteins, collecting physical measurements, or relying on the pool of natural protein variants.

Assessment of antibody library diversity through next generation sequencing and technical error compensation.

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.

Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies.

The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

ABSENCE OF A THYROID PHENOTYPE IN SORTILIN-DEFICIENT MICE.

OBJECTIVE: The Vps10p family member sortilin is expressed in thyroid epithelial cells where it contributes to recycling of the thyroid hormone precursor thyroglobulin (Tg), a process that is thought to render hormone release more effective. Here we investigated the functional impact of sortilin in the thyroid gland using sortilin-deficient mice. METHODS: We measured free T4, thyroid-stimulating hormone (TSH) and Tg serum levels and studied thyroid morphology in 14 sortilin-deficient (Sort1)(-/-)and 12 wildtype (WT) mice. RESULTS: Serum free T4 levels did not differ between Sort1(-/-)and WT females but were significantly lower in Sort1(-/-)males compared with WT (P = .0424). Neither serum TSH nor Tg levels differed between Sort1(-/-)and WT mice, regardless of sex. On the same line, no thyroid histology differences were observed. CONCLUSION: Our findings seem to exclude a role of sortilin in thyroid hormone secretion, although it is possible that the absence of sortilin may result in a thyroid phenotype if combined with other molecular defects of thyroid hormone synthesis and secretion or under iodine deficiency.

The bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1) provides long-term survival in a murine glioma model.

BACKGROUND: Glioblastomas are largely unresponsive to all available treatments and there is therefore an urgent need for novel therapeutics. Here we have probed the antineoplastic effects of a bacterial protein toxin, the cytotoxic necrotizing factor 1 (CNF1), in the syngenic GL261 glioma cell model. CNF1 produces a long-lasting activation of Rho GTPases, with consequent blockade of cytodieresis in proliferating cells and promotion of neuron health and plasticity. METHODS: We have tested the antiproliferative effects of CNF1 on GL261 cells and human glioma cells obtained from surgical specimens. For the in vivo experiments, we injected GL261 cells into the adult mouse visual cortex, and five days later we administered either a single intracerebral dose of CNF1 or vehicle. To compare CNF1 with a canonical antitumoral drug, we infused temozolomide (TMZ) via minipumps for 1 week in an additional animal group. RESULTS: In culture, CNF1 was very effective in blocking proliferation of GL261 cells, leading them to multinucleation, senescence and death within 15 days. CNF1 had a similar cytotoxic effect in primary human glioma cells. CNF1 also inhibited motility of GL261 cells in a scratch-wound migration assay. Low dose (2 nM) CNF1 and continuous TMZ infusion significantly prolonged animal survival (median survival 35 days vs. 28 days in vehicle controls). Remarkably, increasing CNF1 concentration to 80 nM resulted in a dramatic enhancement of survival with no obvious toxicity. Indeed, 57% of the CNF1-treated animals survived up to 60 days following GL261 glioma cell transplant. CONCLUSIONS: The activation of Rho GTPases by CNF1 represents a novel potential therapeutic strategy for the treatment of central nervous system tumors.

Enalapril reduces proliferation and hyaluronic acid release in orbital fibroblasts.

BACKGROUND: Orbital fibroblast proliferation and hyaluronic acid (HA) release are responsible for some of the clinical features of Graves' ophthalmopathy (GO). Thus, inhibition of these processes may be a possible therapeutic approach to this syndrome. Enalapril, a widely used antihypertensive drug, was found to have some inhibitory actions on fibroblast proliferation in cheloid scars in vivo, based on which we investigated its effects in primary cultures of orbital fibroblasts from GO patients and control subjects. METHODS: Primary cultures of GO and control fibroblasts were treated with enalapril or with a control compound (lisinopril). Cell proliferation assays, lactate dehydrogenase release assays (as a measure of cell necrosis), apoptosis assays, and measurement of HA in the cell media were performed. RESULTS: Enalapril significantly reduced cell proliferation in both GO and control fibroblasts. Because enalapril did not affect cell necrosis and apoptosis, we concluded that its effects on proliferation reflected an inhibition of cell growth and/or a delay in cell cycle. Enalapril significantly reduced HA concentrations in the media from both GO and control fibroblasts. CONCLUSIONS: Enalapril has antiproliferative and HA suppressing actions in both GO and control fibroblasts. Clinical studies are needed to investigate whether enalapril has any effects in vivo in patients with GO.

Haptoglobin deficiency determines changes in adipocyte size and adipogenesis.

Haptoglobin (Hp) is an inflammatory and adiposity marker, its expression during obesity being specifically induced in the white adipose tissue (WAT). We previously reported that when challenged with a high fat diet (HFD) Hp-/- mice are partially protected from the onset of insulin resistance and hepatosteatosis. The aim of the present study was to get further insights into Hp function in WAT. To this end, we performed histological and gene expression analysis of the Hp-/- WAT, both in standard and obesity conditions, and investigated how Hp deficiency impacts adipogenesis and WAT development. The average size and percentage of very large adipocytes were respectively smaller and reduced in HFD Hp-/- mice as compared with HFD WT. The expression of perilipin, HSL and angiogenesis related markers were increased in HFD Hp-/- mice. Lean adult Hp-/- showed significantly larger adipocytes and lower subcutaneous WAT expression of aP2 and LPL with respect to WT. Hp-/- young mice (P30) were characterized by larger adipocyte size and lower expression of adipocyte and adipogenesis markers. Comparison of adipocyte size distribution between young and adult mice revealed attenuated changes in Hp-/- mice compared with WT. Mouse embryonic fibroblasts from Hp-/- mice were less capable of accumulating triglycerides and exhibited lower expression of PPARγ, aP2, FAS, LPL and Leptin. In conclusion, Hp deficiency tends to blunt the effect of age and diet on the size of adipocytes, which show less susceptibility to develop hypertrophy during obesity and a reduced adipogenic/hyperplastic potential during youth. In addition, Hp deficiency impacts negatively on adipogenesis.

Human leptin tissue distribution, but not weight loss-dependent change in expression, is associated with methylation of its promoter.

Leptin is a master regulator of energy homeostasis. Its expression, prevalently localized in adipocytes, is positively related to adipose mass. Epigenetics is emerging as an important contributor to the changes in gene expression undergone by adipose tissue during obesity. We herein investigated the involvement of methylation-dependent mechanisms in leptin regulation in humans. We studied the methylation profile of a 305 bp region in the leptin promoter and analyzed the correspondent leptin expression in visceral adipocyte fraction (AF) and stromal vascular fraction (SVF) of white adipose tissue (WAT) and liver. We found an inverse relationship between methylation and leptin expression with AF displaying a lower methylation density (8%) than SVF and liver (18%, 21%). We evidenced a hot spot region, which mostly differentiates AF versus liver. This includes C15 and 21, which are within the recognition sequences for the transcription factors Sp1 and C/EBP, and C22-23/24, flanking a TATA box. In vitro studies demonstrated that demethylation (by decitabine) increase or de novo activate leptin expression in primary fibroblasts and HeLa cells, respectively. A longitudinal study carried out in patients analyzed before and after bariatric surgery-induced weight loss indicated that in this case decrease in WAT leptin expression (about 50%) does not correspond to changes in promoter methylation density. In conclusion, methylation density in the leptin promoter constitutes one control level for cell type specific leptin expression, whereas weight-loss induced changes in leptin expression does not seem to be methylation-dependent.

Obesity-associated hepatosteatosis and impairment of glucose homeostasis are attenuated by haptoglobin deficiency.

OBJECTIVE: Haptoglobin (Hp) is upregulated in both inflammation and obesity. The low chronic inflammatory state, caused by massive adipose tissue macrophage (ATM) infiltration found in obesity, and low adiponectin have been implicated in the development of insulin resistance and hepatosteatosis. The aim of this work was to investigate whether and how Hp interferes with the onset of obesity-associated complications. RESEARCH DESIGN AND METHODS: Hp-null (Hp(-/-)) and wild-type (WT) mice were metabolically profiled under chow-food diet (CFD) and high-fat diet (HFD) feeding by assessing physical parameters, glucose tolerance, insulin sensitivity, insulin response to glucose load, liver triglyceride content, plasma levels of leptin, insulin, glucose, and adiponectin. ATM content was evaluated by using immunohistochemistry (anti-F4/80 antibody). Adiponectin expression was measured in Hp-treated, cultured 3T3-L1 and human adipocytes. RESULTS: No genotype-related difference was found in CFD animals. HFD-Hp(-/-) mice revealed significantly higher glucose tolerance, insulin sensitivity, glucose-stimulated insulin secretion, and adiponectin expression and reduced hepatomegaly/steatosis compared with HFD-WT mice. White adipose tissue (WAT) of HFD-Hp(-/-) mice showed higher activation of insulin signaling cascade, lower ATM, and higher adiponectin expression. Hp was able to inhibit adiponectin expression in cultured adipocytes. CONCLUSIONS: We demonstrated that in the absence of Hp, obesity-associated insulin resistance and hepatosteatosis are attenuated, which is associated with reduced ATM content, increased plasma adiponectin, and higher WAT insulin sensitivity.

The obesity and inflammatory marker haptoglobin attracts monocytes via interaction with chemokine (C-C motif) receptor 2 (CCR2).

BACKGROUND: Obesity is a chronic low inflammatory state. In the obesity condition the white adipose tissue (WAT) is massively infiltrated with monocytes/macrophages, and the nature of the signals recruiting these inflammatory cells has yet to be fully elucidated. Haptoglobin (Hp) is an inflammatory marker and its expression is induced in the WAT of obese subjects. In an effort to elucidate the biological significance of Hp presence in the WAT and of its upregulation in obesity we formulated the hypothesis that Hp may serve as a macrophage chemoattractant. RESULTS: We demonstrated by chemotaxis assay that Hp is able to attract chemokine (C-C motif) receptor 2 (CCR2)-transfected pre-B lymphocytes and monocytes in a dose-dependent manner. Moreover, Hp-mediated migration of monocytes is impaired by CCR2-specific inhibition or previous cell exposure to monocyte chemoattractant protein 1 (MCP1) (also known as CCR2 ligand or chemokine (C-C motif) ligand 2 (CCL2)). Downstream effects of Hp/CCR2 interaction were also investigated: flow cytometry proved that monocytes treated with Hp show reduced CCR2 expression on their surface; Hp interaction induces calcium release that is reduced upon pretreatment with CCR2 antagonist; extracellular signal-regulated kinase (ERK)1/2, a signal transducer activated by CCR2, is phosphorylated following Hp treatment and this phosphorylation is reduced when cells are pretreated with a specific CCR2 inhibitor. Consistently, blocking the ERK1/2 pathway with U0126, the selective inhibitor of the ERK upstream mitogen-activated protein (MAP)-ERK kinase (MEK), results in a dramatic reduction (by almost 100%) of the capability of Hp to induce monocyte migration. CONCLUSIONS: Our data show that Hp is a novel monocyte chemoattractant and that its chemotactic potential is mediated, at least in part. by its interaction with CCR2.

Sortilin is a putative postendocytic receptor of thyroglobulin.

The Vps10p family member sortilin is involved in various cell processes, including protein trafficking. Here we found that sortilin is expressed in thyroid epithelial cells (thyrocytes) in a TSH-dependent manner, that the hormone precursor thyroglobulin (Tg) is a high-affinity sortilin ligand, and that binding to sortilin occurs after Tg endocytosis, resulting in Tg recycling. Sortilin was found to be expressed intracellularly in thyrocytes, as observed in mouse, human, and rat thyroid as well as in FRTL-5 cells. Sortilin expression was demonstrated to be TSH dependent, both in FRTL-5 cells and in mice treated with methimazole and perchlorate. Plasmon resonance binding assays showed that Tg binds to sortilin in a concentration-dependent manner and with high affinity, with Kd values that paralleled the hormone content of Tg. In addition, we found that Tg and sortilin interact in vivo and in cultured cells, as observed by immunoprecipitation, in mouse thyroid extracts and in COS-7 cells transiently cotransfected with sortilin and Tg. After incubation of FRTL-5 cells with exogenous, labeled Tg, sortilin and Tg interacted intracellularly, presumably within the endocytic pathway, as observed by immunofluorescence and immunoelectron microscopy, the latter technique showing some degree of Tg recycling. This was confirmed in FRTL-5 cells in which Tg recycling was reduced by silencing of the sortilin gene and in CHO cells transfected with sortilin in which recycling was increased. Our findings provide a novel pathway of Tg trafficking and a novel function of sortilin in the thyroid gland, the functional impact of which remains to be established.

TSH-Dependent expression of the LDL receptor-associated protein (RAP) in thyroid epithelial cells.

The low density lipoprotein (LDL) receptor-associated protein (RAP) is an endoplasmic reticulum (ER)-resident molecular chaperone for several LDL receptor family members and it also binds to thyroglobulin (Tg), the thyroid hormone precursor. Disruption of the RAP gene in thyrocytes results in impaired Tg secretion. To gain further insights into the function of RAP in the thyroid, we investigated whether its expression in thyrocytes is regulated by thyroid-stimulating hormone (TSH), a feature common to all proteins involved in thyroid hormone secretion. We found by immunofluorescence that in FRTL-5 cells cultured in the presence of TSH, RAP is expressed intracellularly. The levels of expression increased after exposure to TSH, beginning at 48 hours, in a concentration-dependent manner as observed by immunofluorescence and Western blotting. Expression of RAP was also increased by TSH in primary cultures of human thyrocytes as observed by Western blotting. In hypothyroid mice with high serum TSH, RAP was markedly increased compared with euthyroid mice as observed by immunohistochemistry and Western blotting. Based on these findings, we concluded that RAP is expressed by thyrocytes in a TSH-dependent manner, both in cultured thyroid cells and in vivo.

ClC-5 does not affect megalin expression and function in the thyroid.

Megalin is an endocytic receptor responsible for thyroglobulin (Tg) transcytosis, a process that favors hormone release. Accordingly, megalin KO mice have primary hypothyroidism. In the kidney, megalin expression is reduced when the gene encoding the chloride transporter ClC-5 is mutated. We investigated whether megalin expression and function in the thyroid are affected by ClC-5 using a ClC-5 KO mouse model. By Western blotting, ClC-5 was found in thyroid tissue extracts of WT, but not of ClC-5 KO mice. In addition, ClC-5 was found to be expressed by cultured thyroid cells (FRTL-5). The thyroid size, weight, and histology were similar in ClC- 5 KO and WT mice, as were the amounts of megalin in thyroid extracts. Accordingly, serum Tg, a measure of megalin-mediated transcytosis, was similar in WT and ClC-5 KO mice, suggesting that megalin function was unaffected. Thus, unlike in megalin KO mice, in ClC-5 KO mice thyroid function was unchanged, as indicated by the normal serum FT4 and TSH. We concluded that in the thyroid, unlike in the kidney, ClC-5 does not affect megalin expression and function, suggesting that megalin is differentially regulated in these two organs.

Defective thyroglobulin storage in LDL receptor-associated protein-deficient mice.

The molecular chaperone receptor-associated protein (RAP) is required for biosynthesis of megalin, an endocytic receptor for follicular thyroglobulin (Tg), the thyroid hormone precursor. RAP also binds to Tg itself, suggesting that it may affect Tg trafficking in various manners. To elucidate RAP function, we have studied the thyroid phenotype in RAP-knockout (RAP-KO) mice and found a reduction of Tg aggregates into thyroid follicles. Serum Tg levels were significantly increased compared with those of wild-type (WT) mice, suggesting a directional alteration of Tg secretion. In spite of these abnormalities, hormone secretion was maintained as indicated by normal serum thyroxine levels. Because Tg in thyroid extracts from RAP-KO mice contained thyroxine residues as in WT mice, we concluded that in RAP-KO mice, follicular Tg, although reduced, was nevertheless sufficient to provide normal hormone secretion. Serum TSH was increased in RAP-KO mice, and although no thyroid enlargement was observed, some histological features resembling early goiter were present. Megalin was decreased in RAP-KO mice, but this did not affect thyroid function, probably because of the concomitant reduction of follicular Tg. In conclusion, RAP is required for the establishment of Tg reservoirs, but its absence does not affect hormone secretion.

Thyroid dysfunction in megalin deficient mice.

Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.

RET/PTC3 rearrangement and thyroid differentiation gene analysis in a struma ovarii fortuitously revealed by elevated serum thyroglobulin concentration.

Struma ovarii (SO) is usually asymptomatic and only in a few cases it is associated with thyrotoxicosis. The presurgical diagnosis is very uncommon. In the majority of cases a pelvic mass is discovered at physical examination or by abdominal ultrasound. Only the hystopathologic examination is able to reveal the characteristic features of SO, with thyroid cells organized in follicles as the main tumoral tissue constituent. The histologic recognition of malignancy is not easy and usually requires an exhaustive sampling of the lesion to evaluate the extracapsular invasion. We report the case of a 59-year-old woman who came to our observation for the fortuitous finding of elevated serum thyroglobulin (Tg) levels (600-800 ng/mL). Because the thyroid function was normal and the ultrasound showed only a subcentrimetric nodule, the clinical suspicious of a SO was considered. Ultrasound examination of the abdomen showed a solid mass of 2 cm in the left ovary. A (131)I uptake was observed at scintiscan in the site of the solid mass. Three months after the resection of the left ovary serum Tg levels were markedly reduced (106 ng/mL), and its values continued to decrease down to 34 ng/mL at last control. The histology showed that the ovarian mass was mainly constituted of thyroid tissue (98%), with no malignant features. The molecular analysis of several thyroid differentiation gene mRNAs in the SO tissue showed an abundant expression of all genes but pendrin (PDS). A reduced PDS mRNA expression might explain the defective thyroxine (T(4)) production. Despite the absence of malignant features, the expression of RET/PTC3 rearrangement was found, raising the possibility of a potential malignant nature of the tumor. A cancer-free period of 3-4 years, as in our patient, is not long enough to definitively exclude a late onset metastatic disease but, unfortunately, the patient died of nonmedical reasons. In conclusion, we report a case of SO that, to our knowledge, is the first in which the clinical suspicion arose from the inappropriately elevated presurgical serum levels of Tg. A quite exhaustive molecular analysis of thyroid specific genes and oncogenes provided two interesting findings: the low PDS mRNA expression, which may explain the low hormonal production and the absence of thyrotoxicosis and the presence of a RET/PTC3 rearrangement, which prompts the possibility of a late malignant evolution.

Dissection of DIAP1 functional domains via a mutant replacement strategy.

Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of active caspases. Drosophila IAP1 (DIAP1) activity is required to keep cells from undergoing apoptosis. The central cell death regulators Reaper and Hid induce apoptosis very rapidly by inhibiting DIAP1 function. We have developed a system for replacing endogenous DIAP1 with mutant forms of the protein, allowing us to examine the roles of various domains of the protein in living and dying cells. We found that DIAP1 is cleaved by a caspase early after the initiation of apoptosis. This cleavage is required for DIAP1 degradation, but Rpr and Hid can still initiate apoptosis in the absence of cleavage. The cleavage of DIAP1 promotes DIAP1 degradation in a manner dependent on the function of the ubiquitin ligase function of the DIAP1 ring domain. This ring domain function is required for Hid-induced apoptosis. We propose a model that synthesizes our data with those of other laboratories and provide a consistent model for DIAP1 function in living and dying cells.

Genetic screening for melanocortin-4 receptor mutations in a cohort of Italian obese patients: description and functional characterization of a novel mutation.

Mutations in the human melanocortin-4 receptor (MC4-R) gene may account for up to 5.8% of morbid nonsyndromic obesity. We have screened 120 unrelated obese patients for variants of the MC4-R gene. Four heterozygous missense variants were detected, including two polymorphisms (Val(103)Ile and Ile(251)Leu) previously described in the literature. A novel heterozygous mutation (Glu(308)Lys) was detected in a 36-yr-old female patient. Compared with the wild-type receptor, cells expressing the mutated receptor showed a reduced stimulation of cAMP production and a reduction of radioactive alpha MSH binding. No segregation of the mutation with the obese phenotype could be demonstrated. A second, potentially pathogenic mutation (Ser(30)Phe) was detected in a 31-yr-old female patient. Functional analysis of the mutated receptor showed no change in the affinity to the natural ligand alpha MSH nor limited ability to stimulate cAMP production. Sixty lean subjects were also screened, and no additional variants of the MC4-R gene were observed, except for two individuals with the Val(103)Ile polymorphism. In conclusion, we have screened a population of Italian obese subjects for MC4-R variants, demonstrating a 1.7% prevalence of potentially pathogenic mutations. A novel heterozygous missense mutation (Glu(308)Lys) that impairs MC4-R functional activity in vitro was characterized.