on PloS one
by Fusco C, Micale L, Egorov M, Monti M, D'Addetta EV, Augello B, Cozzolino F, Calcagnì A, Fontana A, Polishchuk RS, Didelot G, Reymond A, Pucci P, Merla G
In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.
on The FEBS journal
by Troise F, Monti M, Merlino A, Cozzolino F, Fedele C, Russo Krauss I, Sica F, Pucci P, D'Alessio G, De Lorenzo C
Two novel human antitumor immunoconjugates, engineered by fusion of a single-chain antibody fragment against human ErbB2 receptor, termed Erbicin, with either a human RNase or the Fc region of a human IgG(1) , are selectively cytotoxic for ErbB2-positive cancer cells in vitro and in vivo. These Erbicin-derived immunoagents (EDIAs) do not show the most negative properties of Herceptin, the only humanized mAb against ErbB2 used in the therapy of breast carcinoma: cardiotoxicity and the inability to act on resistant tumors. These differences are probably attributable to the different ErbB2 epitopes recognized by EDIAs and Herceptin, respectively, as we have previously reported that they induce different signaling mechanisms that control tumor and cardiac cell viability. Thus, to accurately identify the novel epitope recognized by EDIAs, three independent and complementary methodologies were used. They gave coherent results, which are reported here: EDIAs bind to a different ErbB2 epitope than Herceptin and the other human/humanized antibodies against ErbB2 reported so far. The epitope has been successfully located in region 122-195 of extracellular domain I. These findings could lead to the identification of novel epitopes on ErbB2 that could be used as potential therapeutic targets to mitigate anti-ErbB2-associated cardiotoxicity and eventually overcome resistance.
on Journal of cellular physiology
by Zanca C, Cozzolino F, Quintavalle C, Di Costanzo S, Ricci-Vitiani L, Santoriello M, Monti M, Pucci P, Condorelli G
PED (phosphoprotein enriched in diabetes) is a 15 kDa protein involved in many cellular pathways and human diseases including type II diabetes and cancer. We recently reported that PED is overexpressed in human cancers and mediates resistance to induced apoptosis. To better understand its role in cancer, we investigated on PED interactome in non-small cell lung cancer (NSCLC). By the Tandem Affinity Purification (TAP), we identified and characterized among others, Rac1, a member of mammalian Rho GTPase protein family, as PED-interacting protein. In this study we show that PED coadiuvates Rac1 activation by regulating AKT mediated Rac1-Ser(71) phosphorylation. Furthermore, we show that the expression of a constitutively active Rac, affected PED-Ser(104) phosphorylation, which is important for PED-regulated ERK 1/2 nuclear localization. Through specific Rac1-siRNA or its pharmacological inhibition, we demonstrate that PED augments migration and invasion in a Rac1-dependent manner in NSCLC. In conclusion, we show for the first time that PED and Rac1 interact and that this interaction modulates cell migration/invasion processes in cancer cells through ERK1/2 pathway.
on Cancer research
by Landriscina M, Laudiero G, Maddalena F, Amoroso MR, Piscazzi A, Cozzolino F, Monti M, Garbi C, Fersini A, Pucci P, Esposito F
TRAP1, a mitochondrial chaperone (Hsp75) with antioxidant and antiapoptotic functions, is involved in multidrug resistance in human colorectal carcinoma cells. Through a proteomic analysis of TRAP1 coimmunoprecipitation complexes, the Ca(2+)-binding protein Sorcin was identified as a new TRAP1 interactor. This result prompted us to investigate the presence and role of Sorcin in mitochondria from human colon carcinoma cells. Using fluorescence microscopy and Western blot analysis of purified mitochondria and submitochondrial fractions, we showed the mitochondrial localization of an isoform of Sorcin with an electrophoretic motility lower than 20 kDa that specifically interacts with TRAP1. Furthermore, the effects of overexpressing or downregulating Sorcin and/or TRAP1 allowed us to demonstrate a reciprocal regulation between these two proteins and to show that their interaction is required for Sorcin mitochondrial localization and TRAP1 stability. Indeed, the depletion of TRAP1 by short hairpin RNA in colorectal carcinoma cells lowered Sorcin levels in mitochondria, whereas the depletion of Sorcin by small interfering RNA increased TRAP1 degradation. We also report several lines of evidence suggesting that intramitochondrial Sorcin plays a role in TRAP1 cytoprotection. Finally, preliminary evidence that TRAP1 and Sorcin are both implicated in multidrug resistance and are coupregulated in human colorectal carcinomas is provided. These novel findings highlight a new role for Sorcin, suggesting that some of its previously reported cytoprotective functions may be explained by involvement in mitochondrial metabolism through the TRAP1 pathway.
on European biophysics journal : EBJ
by Monti DM, Guglielmi F, Monti M, Cozzolino F, Torrassa S, Relini A, Pucci P, Arciello A, Piccoli R
In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an alpha-helical state, which undergoes aggregation at 37 degrees C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of alpha-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.
on Cancer research
by Federico A, Pallante P, Bianco M, Ferraro A, Esposito F, Monti M, Cozzolino M, Keller S, Fedele M, Leone V, Troncone G, Chiariotti L, Pucci P, Fusco A
Chromobox protein homologue 7 (CBX7) is a chromobox family protein encoding a novel polycomb protein, the expression of which shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular, and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins. Among these proteins, we selected histone deacetylase 2 (HDAC2), which is well known to play a key role in neoplastic cell transformation and down-regulation of E-cadherin expression, the loss of which is a critical event in the epithelial-to-mesenchymal transition. We confirmed by coimmunoprecipitation that CBX7 physically interacts with the HDAC2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter and that CBX7 up-regulates E-cadherin expression. Consistent with these data, we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we showed that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype.
on Expert review of proteomics
by Monti M, Cozzolino M, Cozzolino F, Vitiello G, Tedesco R, Flagiello A, Pucci P
Complete description of the complex network of cellular mechanisms and use of the network to predict the full range of cellular behaviors are major goals of systems biology. A key role in contemporary biology can be played by functional proteomics, which focuses on the elucidation of protein functions and the definition of cellular mechanisms at the molecular level. The attainment of these targets is strictly dependent on the identification of individual proteins within functional complexes in vivo. Isolation of interacting proteins relies on either affinity-based or immunoprecipitation procedures in which the protein bait and its specific partners can be fished out by their specific binding to ligand molecules immobilized on insoluble supports. These approaches led to the final identification of several proteins belonging to distinct complexes endowed with different biological functions. Assignment of each protein to a specific complex constitutes a tremendous problem that can only be partially solved using protein-protein interaction databases and literature information. The development of prefractionation methodologies to separate individual protein complexes while preserving their native interactions might then represent an essential tool for the future of functional proteomics. Prepurification of single complexes can only be pursued under native conditions on the basis of their physicochemical features, such as size, dimension (gel filtration chromatography) and density (gradient ultracentrifugation). Following prefractionation, the complex associated to a specific biological function can be isolated using affinity purification techniques. Functional proteomics approaches able to describe individual proteins belonging to complexes involved in specific cellular functions will have a terrific impact on future systems biology studies.
by Pisa V, Cozzolino M, Gargiulo S, Ottone C, Piccioni F, Monti M, Gigliotti S, Talamo F, Graziani F, Pucci P, Verrotti AC
In metazoa, the spatio-temporal translation of diverse mRNAs is essential to guarantee proper oocyte maturation and early embryogenesis. The eukaryotic translation initiation factor 4E (eIF4E), which binds the 5' cap structure of eukaryotic mRNAs, associates with either stimulatory or inhibitory factors to modulate protein synthesis. In order to identify novel factors that might act at the translational level during Drosophila oogenesis, we have undertaken a functional proteomic approach and isolated the product of the Hsp83 gene, the evolutionarily conserved chaperone Hsp90, as a specific component of the cap-binding complex. Here we report that Hsp90 interacts in vitro with the translational repressor Cup. In addition, we show that Hsp83 and cup interact genetically, since lowering Hsp90 activity enhances the oogenesis alterations linked to diverse cup mutant alleles. Hsp90 and Cup co-localize in the cytoplasm of the developing germ-line cells within the germarium, thus suggesting a common function from the earliest stages of oogenesis. Taken together, our data start elucidating the role of Hsp90 during Drosophila female germ-line development and strengthen the idea that Cup has multiple essential functions during egg chamber development.
on Human molecular genetics
by Fraldi A, Zito E, Annunziata F, Lombardi A, Cozzolino M, Monti M, Spampanato C, Ballabio A, Pucci P, Sitia R, Cosma MP
Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion.
on The Italian journal of biochemistry
by Monti M, Cozzolino M, Cozzolino F, Tedesco R, Pucci P
Functional proteomics constitutes an emerging research area in the proteomic field focused to two major targets, the elucidation of biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. Understanding protein functions as well as unravelling molecular mechanisms within the cell is then depending on the identification of the interacting protein partners. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism would in fact be strongly suggestive of its biological function. Furthermore, a detailed description of the cellular signalling pathways might greatly benefit from the elucidation of protein-protein interactions in the cell. Isolation of functional protein complexes essentially rely on affinity-based procedures. The protein of interest and its specific partners can be fished out from the cellular extract by using a suitable ligand as a bait taking advantage of the specific binding properties of the ligand molecule immobilised on agarose-sepharose supports. Alternative strategies essentially relying on immunoprecipitation techniques have been introduced to allow purification of protein complexes formed in vivo within the cell. The gene coding for the bait tagged with an epitope against which good antibodies exist (FLAG, HA, c-myc, etc.), is transfected into the appropriate cell line and expressed in the cognate host. The cell extracts are immunoprecipitated with anti-tag monoclonal antibodies using suitable experimental conditions to avoid dissociation of the complexes. In both cases, protein components specifically recognised by the bait and retained on the agarose beads can then be eluted and fractionated by SDS-PAGE. The protein bands detected on the gel are in situ enzymatically digested and the resulting peptide mixtures analysed by capillary LC-MS/MS techniques leading to the identification of the protein interactors.