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Biomedical subjects

Gerry Melino

Publications and source records attributed to Gerry Melino.

At least 19 recordsLinked to original sources
PubMed2024

Tumor specimen cold ischemia time impacts molecular cancer drug target discovery.

Tumor tissue collections are used to uncover pathways associated with disease outcomes that can also serve as targets for cancer treatment, ideally by comparing the molecular properties of cancer tissues to matching normal tissues. The quality of such collections determines the value of the data and information generated from their analyses including expression and modifications of nucleic acids and proteins. These biomolecules are dysregulated upon ischemia and decompose once the living cells start to decay into inanimate matter. Therefore, ischemia time before final tissue preservation is the most important determinant of the quality of a tissue collection. Here we show the impact of ischemia time on tumor and matching adjacent normal tissue samples for mRNAs in 1664, proteins in 1818, and phosphosites in 1800 cases (tumor and matching normal samples) of four solid tumor types (CRC, HCC, LUAD, and LUSC NSCLC subtypes). In CRC, ischemia times exceeding 15 min impacted 12.5% (mRNA), 25% (protein), and 50% (phosphosites) of differentially expressed molecules in tumor versus normal tissues. This hypoxia- and decay-induced dysregulation increased with longer ischemia times and was observed across tumor types. Interestingly, the proteomics analysis revealed that specimen ischemia time above 15 min is mostly associated with a dysregulation of proteins in the immune-response pathway and less so with metabolic processes. We conclude that ischemia time is a crucial quality parameter for tissue collections used for target discovery and validation in cancer research.

Humans↗
PubMed2006

Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells.

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

Animals↗
PubMed2006

p63 is upstream of IKK alpha in epidermal development.

The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (DeltaNp63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKKalpha, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKKalpha and Ets-1, respectively. We also found that DeltaNp63 upregulates IKKalpha indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKKalpha in epithelial development.

Animals↗
PubMed2006

A cytoplasmic PML mutant inhibits p53 function.

The promyelocytic leukaemia gene (Pml) is a tumor suppressor identified in acute promyelocytic leukaemia (APL), where it is fused to RAR alpha gene as a result of the chromosomal translocation t(15;17). Pml encodes both nuclear and cytoplasmic isoforms. While nuclear PML has been intensively investigated, cytoplasmic PML proteins are less characterized. PML nuclear isoforms (nPML) are the essential components of subnuclear structures referred to as PML nuclear bodies (PML-NB). In response to cellular insults such as DNA damage and oncogenic activation, nPML modulates p53 activity through CBP-mediated acetylation and activates its pro-apoptotic and growth suppressive functions. Two missense mutations resulting in truncated PML cytoplasmic proteins (Mut PML) have been identified in aggressive APL cases. Here we report that cytoplasmic PML is able to induce the relocation of nPML to the cytoplasm, thus reducing the number of PML-NBs. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive, and apoptotic functions, thus suggesting that cytoplasmic expression of PML has an impact on survival through inhibition of nuclear PML. Overall our findings shed new light on the role of PML cytoplasmic proteins in the regulation of p53.

Apoptosis↗
PubMed2006

New p63 targets in keratinocytes identified by a genome-wide approach.

p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The DeltaNp63alpha isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DeltaNp63alpha; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

Animals↗
PubMed2006

p63 and p73, members of the p53 gene family, transactivate PKCdelta.

The p53 family comprises three genes that encode for p53, p63 and p73. These genes have a significant degree of sequence homology, especially in the central sequence-specific DNA-binding domain. The high homology among the three DNA-binding domains indicates that these transcription factors have identical residues interacting with DNA, and thus potentially can recognize the same transcriptional targets. In this study, we demonstrate that PKCdelta is induced by p63 and p73 in Saos2 cells. The putative human PKCdelta promoter harbours three p53-like binding sites (RE I, RE II, RE III). In order to confirm the transactivation of PKCdelta by p53 family members, we performed transcription assays using the entire or selected regions of the promoter upstream of a luciferase reporter gene. The results obtained demonstrated that, at least in vitro, the p53 family members tested (TAp63alpha, TAp73alpha, DeltaNp63alpha, but not DeltaNp73alpha) were able to drive transcription from the PKCdelta promoter.

Cell Line, Tumor↗
PubMed2006

The E3 ubiquitin ligase Itch controls the protein stability of p63.

p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile alpha motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions.

Animals↗
PubMed2006

TAp73alpha induces tau phosphorylation in HEK293a cells via a transcription-dependent mechanism.

p73 and tau both play roles in neurodevelopment and neurodegeneration. In this pilot study we show by Western blotting that TAp73alpha induces phosphorylation of human 2N4R tau at threonine-205 and at the PHF-1 epitope (serine366/serine404) in HEK293a cells. Neither the dominant negative isoform, DeltaNp73, nor a transcriptionally inactive mutant TAp73alpha(R292H) altered tau phosphorylation indicating that tau phosphorylation is dependent on the transcriptional activity of TAp73alpha. Consistent with this, confocal microscopy revealed that tau and TAp73alpha were spatially separated within the cell; tau being located in the cytoskeletal compartment whilst TAp73alpha was found in the nucleus. These findings have ramifications for microtubule dynamics associated with axonal growth during development and for neuronal death associated with Alzheimer's disease and other tauopathies.

Alzheimer Disease↗
PubMed2006

Cytoplasmic function of mutant promyelocytic leukemia (PML) and PML-retinoic acid receptor-alpha.

The promyelocytic leukemia (PML) tumor suppressor of acute promyelocytic leukemia (APL) regulates major apoptotic and growth-suppressive pathways. In APL, PML is involved in a chromosomal translocation generating the PML-retinoic acid receptor-alpha (RARalpha) fusion protein. Two missense mutations in the remaining PML alleles have been identified, which give rise to a truncated cytoplasmic PML protein (Mut PML). APL patients carrying these mutations display resistance to retinoic acid (RA) and very poor prognosis. Here we show that Mut PML associates with the cytoplasmic regions we refer to as PML-cytoplasmic bodies (PML-CBs). Mut PML interacts with PML-RARalpha in PML-CB and potentiates PML-RARalpha-mediated inhibition of RA-dependent transcription. Remarkably, Mut PML stabilizes PML-RARalpha and inhibits differentiation induced by pharmacological doses of RA. A mutant form of PML-RARalpha that accumulates in the cytoplasm inhibits RA-dependent transcription and differentiation, thus suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we show that the bcr3 PML-RARalpha form is predominantly cytoplasmic and accumulates in PML-CBs. Taken together, these findings reveal novel insights into the molecular mechanisms contributing to APL.

Animals↗
PubMed2006

p53 is cleaved by caspases generating fragments localizing to mitochondria.

The p53 tumor suppressor protein exerts most of its anti-tumorigenic activity by transcriptionally activating several pro-apoptotic genes. Accumulating evidence also suggests a transcription-independent function of p53 during apoptosis. It has recently been shown that, when activated, a fraction of p53 translocates to mitochondria, causing cytochrome c release. We now demonstrate a caspase-dependent cleavage of p53 resulting in the generation of four fragments, two of which lack a nuclear localization signal and consequently localize to cytosol. Moreover, these two fragments translocate to mitochondria and induce mitochondrial membrane depolarization in the absence of transcriptional activity. This novel feature of p53 supports the model whereby cytosolic p53 exerts major functions in apoptosis and also suggests the presence of a positive feedback loop in which activated caspases cleave p53 to augment mitochondrial membrane depolarization.

Amino Acid Sequence↗
PubMed2006

TAp73 isoforms antagonize Notch signalling in SH-SY5Y neuroblastomas and in primary neurones.

p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter-gene assays, we demonstrate that C-promoter binding factor-1 (CBF-1)-dependent gene transcription driven by the Notch-1 intracellular domain (N1(ICD)) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH-SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c-Abl or cisplatin treatment also potently antagonized N1(ICD)/CBF-1-dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split-1 (HES-1) protein levels and antagonized the increase in HES-1 protein induced by exogenous N1(ICD) expression. Evidence of a direct physical interaction between N1(ICD) and TAp73alpha was demonstrated by co-immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73alpha binds the N1(ICD) in a region C-terminal of aa 2094. Interestingly, DeltaNp73alpha and TAp73alpha(R292H) also co-purified with N1(ICD), but neither inhibited N1(ICD)/CBF-1-dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73alpha reversed the N1(ICD)-mediated repression of retinoic acid-induced differentiation of SH-SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73alpha on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis.

Basic Helix-Loop-Helix Proteins↗
PubMed2006

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome.

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile alpha-motif (SAM) domain (residues 505-579), a region crucial to explaining the human genetic disease ankyloblepharonectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of freedom of individual residues and discussion of the possible causes for the pathology.

Abnormalities, Multiple↗
PubMed2005

p63 protein is essential for the embryonic development of vibrissae and teeth.

Development of skin appendages strongly depends on epithelial-mesenchymal interactions. One of the genes involved in this process is p63, a member of the p53 family of transcription factors, essential for ectodermal development, as elucidated by the phenotype of p63 knock-out mice. Surprisingly, no information on p63 expression in tooth and hair is yet available. Here, we show p63 expression during teeth and vibrissae morphogenesis in mouse embryos and we also show a correlation with the expression patterns of the epithelial marker keratin 5 and the proliferation marker Ki67. Our results show that p63 colocalizes with both K5 and Ki67 in the epithelium of developing vibrissae, while in teeth p63 is expressed, together with K5, in the undifferentiated ectoderm (enamel organ), and in ameloblasts, a subpopulation of differentiated ectodermal cells. Moreover, p63 expression in tooth seems not to be fully colocalized with nuclear Ki67 expression.

Animals↗
PubMed2005

Neurodevelopment on route p63.

All known members of the p53 gene family, including the two homologs p73 and p63, have multiple biological functions. In neurons, p53 and p73 are known to regulate cell death in the developing and adult nervous system. A report by Jacobs et al. in this issue of Neuron shows that the more ancestral member of this gene family, p63, is an essential proapoptotic protein during neuronal development.

Animals↗
PubMed2005

The promyelocytic leukaemia protein tumour suppressor functions as a transcriptional regulator of p63.

p63 plays unique developmental roles in epidermal morphogenesis, despite its structural similarity with p53. The p63 gene has two distinct promoters, coding for proteins containing an N-terminal transactivation domain (TA isoforms) and for proteins lacking this region (DeltaN isoforms). The full-length transcriptionally active TAp63 isoforms are capable of transactivating the majority of the p53 target promoters thus inducing cell cycle arrest and apoptosis. On the contrary, the DeltaNp63 isoforms seem to counteract the transactivation activities of p53 and TAp63 proteins, thus possibly conferring a proliferative advantage to cancer cells. However, the molecular mechanisms controlling the transcriptional activity of p63 remain largely unclear. Here we present data indicating that (i) the promyelocytic leukaemia protein (PML) physically interacts with p63, (ii) p63 is localized into the PML nuclear-bodies (PML-NBs) in vivo, and (iii) PML regulates p63 transcriptional activity. We show that the interaction of p63 with PML increases the levels of p63 in cultured cells as well as its ability to transactivate the p53-responsive elements of the GADD45, p21 and bax promoters. These data are consistent with a general role for PML as a functional modulator of all the p53 family members. Our findings strengthen the relevance of the cross talk between PML and the p53 family members, imply a new tumour suppressive function of PML and unveil a possible role for PML in epidermal morphogenesis and differentiation.

DNA-Binding Proteins↗
PubMed2005

A homozygous missense mutation in TGM5 abolishes epidermal transglutaminase 5 activity and causes acral peeling skin syndrome.

Peeling skin syndrome is an autosomal recessive genodermatosis characterized by the shedding of the outer epidermis. In the acral form, the dorsa of the hands and feet are predominantly affected. Ultrastructural analysis has revealed tissue separation at the junction between the granular cells and the stratum corneum in the outer epidermis. Genomewide linkage analysis in a consanguineous Dutch kindred mapped the gene to 15q15.2 in the interval between markers D15S1040 and D15S1016. Two homozygous missense mutations, T109M and G113C, were found in TGM5, which encodes transglutaminase 5 (TG5), in all affected persons in two unrelated families. The mutation was present on the same haplotype in both kindreds, indicating a probable ancestral mutation. TG5 is strongly expressed in the epidermal granular cells, where it cross-links a variety of structural proteins in the terminal differentiation of the epidermis to form the cornified cell envelope. An established, in vitro, biochemical cross-linking assay revealed that, although T109M is not pathogenic, G113C completely abolishes TG5 activity. Three-dimensional modeling of TG5 showed that G113C lies close to the catalytic domain, and, furthermore, that this glycine residue is conserved in all known transglutaminases, which is consistent with pathogenicity. Other families with more-widespread peeling skin phenotypes lacked TGM5 mutations. This study identifies the first causative gene in this heterogeneous group of skin disorders and demonstrates that the protein cross-linking function performed by TG5 is vital for maintaining cell-cell adhesion between the outermost layers of the epidermis.

Binding Sites↗
PubMed2005

Calpain cleavage regulates the protein stability of p73.

The function of p73, a transcription factor belonging to the p53 family, is finely regulated by its steady-state protein stability. p73 protein degradation/stabilization can be regulated by mechanisms in part dependent on the ubiquitin proteasome system (UPS): (i) Itch/NEDD4-like UPS degradation, (ii) NEDD8 UPS degradation, and (iii) NQO1 20S proteasome-dependent (but ubiquitin-independent) breakdown. Here, we show that, in vitro, Calpain I can cleave p73 at two distinct sites: the first proline-rich region and within the oligomerization domain. Consequently, different p73 isoforms can be degraded by calpains, i.e., both N-terminal isoforms (TAp73 and DeltaNp73) as well as the C-terminal isoforms (alpha, beta, gamma, delta). Moreover, overexpression of the specific endogenous calpain inhibitor, calpastatin, in cultured cells increased the steady-state p73 level. This suggests that calpains may play a physiological role in the regulation of p73 protein stability.

Base Sequence↗