Bcl-2 dependent modulation of Hippo pathway in cancer cells.

INTRODUCTION: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Bcl-2 family inhibitors sensitize human cancer models to therapy.

BH3 mimetics, targeting the Bcl-2 family anti-apoptotic proteins, represent a promising therapeutic opportunity in cancers. ABT-199, the first specific Bcl-2 inhibitor, was approved by FDA for the treatment of several hematological malignancies. We have recently discovered IS21, a novel pan BH3 mimetic with preclinical antitumor activity in several tumor types. Here, we evaluated the efficacy of IS21 and other BH3 mimetics, both as single agents and combined with the currently used antineoplastic agents in T-cell acute lymphoblastic leukemia, ovarian cancer, and melanoma. IS21 was found to be active in T-cell acute lymphoblastic leukemia, melanoma, lung, pancreatic, and ovarian cancer cell lines. Bcl-xL and Mcl-1 protein levels predicted IS21 sensitivity in melanoma and ovarian cancer, respectively. Exploring IS21 mechanism of action, we found that IS21 activity depends on the presence of BAX and BAK proteins: complexes between Bcl-2 and Bcl-xL proteins and their main binding partners were reduced after IS21 treatment. In combination experiments, BH3 mimetics sensitized leukemia cells to chemotherapy, ovarian cancer cells and melanoma models to PARP and MAPK inhibitors, respectively. We showed that this enhancing effect was related to the potentiation of the apoptotic pathway, both in hematologic and solid tumors. In conclusion, our data suggest the use of inhibitors of anti-apoptotic proteins as a therapeutic strategy to enhance the efficacy of anticancer treatment.

Bcl-2-like protein-10 increases aggressive features of melanoma cells.

Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis. Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR. Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

Silencing of Ago-2 Interacting Protein SERBP1 Relieves KCC2 Repression by miR-92 in Neurons.

RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (-) extruding K (+) Cl (-) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3' untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3'UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3'UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation.

Targeting the anti-apoptotic Bcl-2 family proteins: machine learning virtual screening and biological evaluation of new small molecules.

Bcl-2 family anti-apoptotic proteins are overexpressed in several hematological and solid tumors, and contribute to tumor formation, progression, and resistance to therapy. They represent a promising therapeutic avenue to explore for cancer treatment. Venetoclax, a Bcl-2 inhibitor is currently used for hematological malignancies or is undergoing clinical trials for either hematological or solid tumors. Despite these progresses, ongoing efforts are focusing on the identification and development of new molecules targeting Bcl-2 protein and/or other family members. Methods: Machine learning guided virtual screening followed by surface plasmon resonance, molecular docking and pharmacokinetic analyses were performed to identify new inhibitors of anti-apoptotic members of Bcl-2 family and their pharmacokinetic profile. The sensitivity of cancer cells from different origin to the identified compounds was evaluated both in in vitro (cell survival, apoptosis, autophagy) and in vivo (tumor growth in nude mice) preclinical models. Results: IS20 and IS21 were identified as potential new lead compounds able to bind Bcl-2, Bcl-xL and Mcl-1 recombinant proteins. Molecular docking investigation indicated IS20 and IS21 could bind into the Beclin-1 BH3 binding site of wild type Bcl-2, Bcl-xL and Mcl-1 proteins. In particular, although the IS21 docked conformation did not show a unique binding mode, it clearly showed its ability in flexibly adapting to either BH3 binding sites. Moreover, both IS20 and IS21 reduced cell viability, clonogenic ability and tumor sphere formation, and induced apoptosis in leukemic, melanoma and lung cancer cells. Autophagosome formation and maturation assays demonstrated induction of autophagic flux after treatment with IS20 or IS21. Experiments with z-VAD-fmk, a pan-caspase inhibitor, and chloroquine, a late-stage autophagy inhibitor, demonstrated the ability of the two compounds to promote apoptosis by autophagy. IS21 also reduced in vivo tumor growth of both human leukemia and melanoma models. Conclusion: Virtual screening coupled with in vitro and in vivo experimental data led to the identification of two new promising inhibitors of anti-apoptotic proteins with good efficacy in the binding to recombinant Bcl-2, Bcl-xL and Mcl-1 proteins, and against different tumor histotypes.

Special Issue "Precision Oncology in Melanoma Progression".

Melanoma represents the most malignant type of skin cancer, with increasing incidence worldwide [...].

Antitumor effect of Melaleuca alternifolia essential oil and its main component terpinen-4-ol in combination with target therapy in melanoma models.

Essential oils (EOs) have been recently emerging for their promising biological activities in preventing tumorigenesis or progression of different tumor histotypes, including melanoma. In this study, we investigated the antitumor activity of a panel of EOs in different tumor models. The ability of Melaleuca alternifolia (tea tree oil) and its main component, terpinen-4-ol, to sensitize the target therapy currently used for melanoma treatment was also assessed. Our results demonstrated that EOs differently affect the viability of human cancer cells and led us to select six EOs effective in melanoma and lung cancer cells, without toxic effects in human fibroblasts. When combined with dabrafenib and/or trametinib, Melaleuca alternifolia synergistically reduced the viability of melanoma cells by activating apoptosis. Through machine learning classification modeling, α-terpineol, tepinolene, and terpinen-4-ol, three components of Melaleuca alternifolia, were identified as the most likely relevant components responsible for the EO's antitumor effect. Among them, terpinen-4-ol was recognized as the Melaleuca alternifolia component responsible for its antitumor and proapoptotic activity. Overall, our study holds promise for further analysis of EOs as new anticancer agents and supports the rationale for their use to improve target therapy response in melanoma.

Hypoxia-dependent drivers of melanoma progression.

Hypoxia, a condition of low oxygen availability, is a hallmark of tumour microenvironment and promotes cancer progression and resistance to therapy. Many studies reported the essential role of hypoxia in regulating invasiveness, angiogenesis, vasculogenic mimicry and response to therapy in melanoma. Melanoma is an aggressive cancer originating from melanocytes located in the skin (cutaneous melanoma), in the uveal tract of the eye (uveal melanoma) or in mucosal membranes (mucosal melanoma). These three subtypes of melanoma represent distinct neoplasms in terms of biology, epidemiology, aetiology, molecular profile and clinical features.In this review, the latest progress in hypoxia-regulated pathways involved in the development and progression of all melanoma subtypes were discussed. We also summarized current knowledge on preclinical studies with drugs targeting Hypoxia-Inducible Factor-1, angiogenesis or vasculogenic mimicry. Finally, we described available evidence on clinical studies investigating the use of Hypoxia-Inducible Factor-1 inhibitors or antiangiogenic drugs, alone or in combination with other strategies, in metastatic and adjuvant settings of cutaneous, uveal and mucosal melanoma.Hypoxia-Inducible Factor-independent pathways have been also reported to regulate melanoma progression, but this issue is beyond the scope of this review.As evident from the numerous studies discussed in this review, the increasing knowledge of hypoxia-regulated pathways in melanoma progression and the promising results obtained from novel antiangiogenic therapies, could offer new perspectives in clinical practice in order to improve survival outcomes of melanoma patients.

SEMAPHORINS and their receptors: focus on the crosstalk between melanoma and hypoxia.

Hypoxia, a condition of oxygen deprivation, is considered a hallmark of tumor microenvironment regulating several pathways and promoting cancer progression and resistance to therapy. Semaphorins, a family of about 20 secreted, transmembrane and GPI-linked glycoproteins, and their cognate receptors (plexins and neuropilins) play a pivotal role in the crosstalk between cancer and stromal cells present in the tumor microenvironment. Many studies reported that some semaphorins are involved in the development of a permissive tumor niche, guiding cell-cell communication and, consequently, the development and progression, as well as the response to therapy, of different cancer histotypes, including melanoma.In this review we will summarize the state of art of semaphorins regulation by hypoxic condition in cancer with different origin. We will also describe evidence about the ability of semaphorins to affect the expression and activity of transcription factors activated by hypoxia, such as hypoxia-inducible factor-1. Finally, we will focus our attention on findings reporting the role of semaphorins in melanocytes transformation, melanoma progression and response to therapy. Further studies are necessary to understand the mechanisms through which semaphorins induce their effect and to shed light on the possibility to use semaphorins or their cognate receptors as prognostic markers and/or therapeutic targets in melanoma or other malignancies.

Inhibition of Anti-Apoptotic Bcl-2 Proteins in Preclinical and Clinical Studies: Current Overview in Cancer.

The dynamic interplay between pro-death and pro-survival Bcl-2 family proteins is responsible for a cell's fate. Due to the recognized relevance of this family in cancer progression and response to therapy, different efforts have made in recent years in order to develop small molecules able to target anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1. The limitations of the first Bcl-2 family targeted drugs, regarding on-target and off-target toxicities, have been overcome with the development of venetoclax (ABT-199), the first BH3 mimetic inhibitor approved by the FDA. The purpose of this review is to discuss the state-of-the-art in the development of drugs targeting Bcl-2 anti-apoptotic proteins and to highlight the potential of their application as single agents or in combination for improving anti-cancer therapy, focusing in particular on solid tumors.

Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages.

BACKGROUND: A bidirectional crosstalk between tumor cells and the surrounding microenvironment contributes to tumor progression and response to therapy. Our previous studies have demonstrated that bcl-2 affects melanoma progression and regulates the tumor microenvironment. The aim of this study was to evaluate whether bcl-2 expression in melanoma cells could influence tumor-promoting functions of tumor-associated macrophages, a major constituent of the tumor microenvironment that affects anticancer immunity favoring tumor progression. METHODS: THP-1 monocytic cells, monocyte-derived macrophages and melanoma cells expressing different levels of bcl-2 protein were used. ELISA, qRT-PCR and Western blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Chromatin immunoprecipitation assay was performed to evaluate transcription factor recruitment at specific promoters. Boyden chamber was used for migration experiments. Cytofluorimetric and immunohistochemical analyses were carried out to evaluate infiltrating macrophages and T cells in melanoma specimens from patients or mice. RESULTS: Higher production of tumor-promoting and chemotactic factors, and M2-polarized activation was observed when macrophages were exposed to culture media from melanoma cells overexpressing bcl-2, while bcl-2 silencing in melanoma cells inhibited the M2 macrophage polarization. In agreement, the number of melanoma-infiltrating macrophages in vivo was increased, in parallel with a greater expression of bcl-2 in tumor cells. Tumor-derived interleukin-1ß has been identified as the effector cytokine of bcl-2-dependent macrophage reprogramming, according to reduced tumor growth, decreased number of M2-polarized tumor-associated macrophages and increased number of infiltrating CD4+IFNγ+ and CD8+IFNγ+ effector T lymphocytes, which we observed in response to in vivo treatment with the IL-1 receptor antagonist kineret. Finally, in tumor specimens from patients with melanoma, high bcl-2 expression correlated with increased infiltration of M2-polarized CD163+ macrophages, hence supporting the clinical relevance of the crosstalk between tumor cells and microenvironment. CONCLUSIONS: Taken together, our results show that melanoma-specific bcl-2 controls an IL-1ß-driven axis of macrophage diversion that establishes tumor microenvironmental conditions favoring melanoma development. Interfering with this pathway might provide novel therapeutic strategies.

microRNA-378a-5p iS a novel positive regulator of melanoma progression.

Evaluating the expression levels of miR-378a-5p both in a large melanoma patient cohort from The Cancer Genome Atlas database and in melanoma patients from our Institute, we found that miR-378a-5p is upregulated in metastatic melanoma specimens. miR-378a-5p expression was also increased in melanoma cells resistant to target therapy, and decreased in response to drug treatment. We also demonstrated that overexpression of miR-378a-5p enhances in vitro cell invasion and migration, and facilitates the ability of melanoma cells to form de novo vasculogenic structures. While performing downstream targeting studies, we confirmed the ability of miR-378a-5p to modulate the expression of known target genes, such as SUFU, FUS-1, and KLF9. Luciferase-3'UTR experiments also identified STAMBP and HOXD10 as new miR-378a-5p target genes. MMP2 and uPAR, two HOXD10 target genes, were positively regulated by miR-378a-5p. Genetic and pharmacologic approaches inhibiting uPAR expression and activity evidenced that the in vitro tumor-promoting functions of miR-378a-5p, were in part mediated by uPAR. Of note miR-378a-5p was also able to increase VEGF, as well as in vitro and in vivo angiogenesis. Finally, genetic and pharmacologic modulation of Bcl-2 evidenced Bcl-2 ability to regulate miR-378a-5p expression. In conclusion, to the best of our knowledge, this is the first study demonstrating that miR-378a-5p acts as an oncogenic microRNA in melanoma.

Semaphorin 5A drives melanoma progression: role of Bcl-2, miR-204 and c-Myb.

BACKGROUND: Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. METHODS: Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. RESULTS: A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. CONCLUSION: Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.

BCL-XL overexpression promotes tumor progression-associated properties.

By using human melanoma and glioblastoma cell lines and their derivative BCL-XL overexpressing clones, we investigated the role of BCL-XL in aggressive features of these two tumor histotypes. We found that in both models, BCL-XL overexpression increased in vitro cell migration and invasion and facilitated tumor cells to form de novo vasculogenic structures. Furthermore, BCL-XL overexpressing cells exhibited higher tumors sphere formation capacity and expressed higher levels of some stem cell markers, supporting the concept that BCL-XL plays essential roles in the maintenance of cancer stem cell phenotype. BCL-XL expression reduction by siRNA, the exposure to a BCL-XL-specific inhibitor and the use of a panel of human melanoma cell lines corroborated the evidence that BCL-XL regulates tumor progression-associated properties. Finally, the vascular markers and the vasculogenic mimicry were up-regulated in the BCL-XL overexpressing xenografts derived from both tumor histotypes. In conclusion, our work brings further support to the understanding of the malignant actions of BCL-XL and, in particular, to the concept that BCL-XL promotes stemness and contributes to the aggressiveness of both melanoma and glioblastoma.

miR-211 and MITF modulation by Bcl-2 protein in melanoma cells.

Melanoma, the most lethal form of skin cancer, is frequently associated with alterations in several genes, among which the Bcl-2 oncogene plays an important role in progression, chemosensitivity and angiogenesis. Also microRNA (miRNA) are emerging as modulators of melanoma development and progression, and among them, miR-211, located within the melastatin-1/TRPM1 (transient receptor potential cation channel, subfamily M, member 1 protein) gene, is prevalently expressed in the melanocyte lineage and acts as oncosuppressor. Using several human melanoma cell lines and their Bcl-2 stably overexpressing derivatives, we evaluated whether there was a correlation between expression of Bcl-2 and miR-211. Western blot analysis and quantitative real-time polymerase chain reaction demonstrated reduced expression of pri-miR-211, miR-211, TRPM1, and MLANA levels, after Bcl-2 overexpression, associated with increased expression of well-known miR-211 target genes. Overexpression of mature miR-211 in Bcl-2 overexpressing cells rescued Bcl-2 ability to increase cell migration. A decreased nuclear localization of microphthalmia-associated transcription factor (MITF), a co-regulator of both miR-211 and TRPM1, and a reduced MITF recruitment at the TRPM1 and MLANA promoters were also evidenced in Bcl-2 overexpressing cells by immunofluorescence and chromatin immunoprecipitation experiments, respectively. Reduction of Bcl-2 expression by small interference RNA confirmed the ability of Bcl-2 to modulate miR-211 and TRPM1 expression. © 2015 Wiley Periodicals, Inc.

Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications.

p63 isoforms regulate metabolism of cancer stem cells.

p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.

On the catalytic mechanism and stereospecificity of Escherichia coli L-threonine aldolase.

L-threonine aldolases (L-TAs) represent a family of homologous pyridoxal 5'-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several L-3-hydroxy-α-amino acids. L-TAs have great biotechnological potential, as they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for the bioorganic synthesis of L-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many L-TAs, showing different stereospecificity towards the Cß configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, L-TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low-specificity L-TA from Escherichia coli at 2.2-Å resolution, in the unliganded form and cocrystallized with L-serine and L-threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro-aldol cleavage reaction. Interestingly, the very large active site opening of E. coli L-TA suggests that much larger molecules than L-threonine isomers may be easily accommodated, and L-TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues.

Shotgun proteomics reveals specific modulated protein patterns in tears of patients with primary open angle glaucoma naïve to therapy.

Primary open angle glaucoma (POAG) is one of the main causes of irreversible blindness worldwide. The pathogenesis of POAG is still unclear. Alteration and sclerosis of trabecular meshwork with changes in aqueous humor molecular composition seem to play the key role. Increased intraocular pressure is widely known to be the main risk factor for the onset and progression of the disease. Unfortunately, the early diagnosis of POAG still remains the main challenge. In order to provide insight into the patho-physiology of glaucoma, here we report a shotgun proteomics approach to tears of patients with POAG naïve to therapy. Our proteomics results showed 27 differential tear proteins in POAG vs. CTRL comparison (25 up regulated proteins in the POAG group and two unique proteins in the CTRL group), 16 of which were associated with inflammatory response, free radical scavenging, cell-to-cell signaling and interaction. Overall the protein modulation shown in POAG tears proves the involvement of biochemical networks linked to inflammation. Among all regulated proteins, a sub-group of 12 up-regulated proteins in naïve POAG patients were found to be down-regulated in medically controlled POAG patients treated with prostanoid analogues (PGA), as reported in our previous work (i.e., lipocalin-1, lysozyme C, lactotransferrin, proline-rich-protein 4, prolactin-inducible protein, zinc-alpha-2-glycoprotein, polymeric immunoglobulin receptor, cystatin S, Ig kappa chain C region, Ig alpha-2 chain C region, immunoglobulin J chain, Ig alpha-1 chain C region). In summary, our findings indicate that the POAG tears protein expression is a mixture of increased inflammatory proteins that could be potential biomarkers of the disease, and their regulation may be involved in the mechanism by which PGA are able to decrease the intraocular pressure in glaucoma patients.