Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

Emerging roles of the cellular prion protein (PrPC) and 37/67 kDa laminin receptor (RPSA) interaction in cancer biology.

The cellular prion protein (PrPC) is well-known for its involvement, under its pathogenic protease-resistant form (PrPSc), in a group of neurodegenerative diseases, known as prion diseases. PrPC is expressed in nervous system, as well as in other peripheral organs, and has been found overexpressed in several types of solid tumors. Notwithstanding, studies in recent years have disclosed an emerging role for PrPC in various cancer associated processes. PrPC has high binding affinity for 37/67 kDa laminin receptor (RPSA), a molecule that acts as a key player in tumorigenesis, affecting cell growth, adhesion, migration, invasion and cell death processes. Recently, we have characterized at cellular level, small molecules able to antagonize the direct PrPC binding to RPSA and their intracellular trafficking. These findings are very crucial considering that the main function of RPSA is to modulate key events in the metastasis cascade. Elucidation of the role played by PrPC/RPSA interaction in regulating tumor development, progression and response to treatment, represents a very promising challenge to gain pathogenetic information and discover novel specific biomarkers and/or therapeutic targets to be exploited in clinical settings. This review attempts to convey a detailed description of the complexity surrounding these multifaceted proteins from the perspective of cancer hallmarks, but with a specific focus on the role of their interaction in the control of proliferation, migration and invasion, genome instability and mutation, as well as resistance to cell death controlled by autophagic pathway.

Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein we identify the molecular mechanisms involved, demonstrating that TRAP1: i) binds both mitochondrial and cytosolic ribosomes as well as translation elongation factors, ii) slows down translation elongation rate, and iii) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

Regulation of mitochondrial complex III activity and assembly by TRAP1 in cancer cells.

BACKGROUND: Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. RESULTS: Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. CONCLUSIONS: These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.

Endosomal trafficking and related genetic underpinnings as a hub in Alzheimer's disease.

Genetic studies support the amyloid cascade as the leading hypothesis for the pathogenesis of Alzheimer's disease (AD). Although significant efforts have been made in untangling the amyloid and other pathological events in AD, ongoing interventions for AD have not been revealed efficacious for slowing down disease progression. Recent advances in the field of genetics have shed light on the etiology of AD, identifying numerous risk genes associated with late-onset AD, including genes related to intracellular endosomal trafficking. Some of the bases for the development of AD may be explained by the recently emerging AD genetic "hubs," which include the processing pathway of amyloid precursor protein and the endocytic pathway. The endosomal genetic hub may represent a common pathway through which many pathological effects can be mediated and novel, alternative biological targets could be identified for therapeutic interventions. The aim of this review is to focus on the genetic and biological aspects of the endosomal compartments related to AD progression. We report recent studies which describe how changes in endosomal genetics impact on functional events, such as the amyloidogenic and non-amyloidogenic processing, degradative pathways, and the importance of receptors related to endocytic trafficking, including the 37/67 kDa laminin-1 receptor ribosomal protein SA, and their implications for neurodegenerative diseases.

Activation of Non-Canonical Autophagic Pathway through Inhibition of Non-Integrin Laminin Receptor in Neuronal Cells.

To fight neurodegenerative diseases, several therapeutic strategies have been proposed that, to date, are either ineffective or at the early preclinical stages. Intracellular protein aggregates represent the cause of about 70% of neurodegenerative disorders, such as Alzheimer's disease. Thus, autophagy, i.e., lysosomal degradation of macromolecules, could be employed in this context as a therapeutic strategy. Searching for a compound that stimulates this process led us to the identification of a 37/67kDa laminin receptor inhibitor, NSC48478. We have analysed the effects of this small molecule on the autophagic process in mouse neuronal cells and found that NSC48478 induces the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) into the LC3-phosphatidylethanolamine conjugate (LC3-II). Interestingly, upon NSC48478 treatment, the contribution of membranes to the autophagic process derived mainly from the non-canonical m-TOR-independent endocytic pathway, involving the Rab proteins that control endocytosis and vesicle recycling. Finally, qRT-PCR analysis suggests that, while the expression of key genes linked to canonical autophagy was unchanged, the main genes related to the positive regulation of endocytosis (pinocytosis and receptor mediated), along with genes regulating vesicle fusion and autolysosomal maturation, were upregulated under NSC48478 conditions. These results strongly suggest that 37/67 kDa inhibitor could be a useful tool for future studies in pathological conditions.

Nuclear FGFR2 Interacts with the MLL-AF4 Oncogenic Chimera and Positively Regulates HOXA9 Gene Expression in t(4;11) Leukemia Cells.

The chromosomal translocation t(4;11) marks an infant acute lymphoblastic leukemia associated with dismal prognosis. This rearrangement leads to the synthesis of the MLL-AF4 chimera, which exerts its oncogenic activity by upregulating transcription of genes involved in hematopoietic differentiation. Crucial for chimera's aberrant activity is the recruitment of the AF4/ENL/P-TEFb protein complex. Interestingly, a molecular interactor of AF4 is fibroblast growth factor receptor 2 (FGFR2). We herein analyze the role of FGFR2 in the context of leukemia using t(4;11) leukemia cell lines. We revealed the interaction between MLL-AF4 and FGFR2 by immunoprecipitation, western blot, and immunofluorescence experiments; we also tested the effects of FGFR2 knockdown, FGFR2 inhibition, and FGFR2 stimulation on the expression of the main MLL-AF4 target genes, i.e., HOXA9 and MEIS1. Our results show that FGFR2 and MLL-AF4 interact in the nucleus of leukemia cells and that FGFR2 knockdown, which is associated with decreased expression of HOXA9 and MEIS1, impairs the binding of MLL-AF4 to the HOXA9 promoter. We also show that stimulation of leukemia cells with FGF2 increases nuclear level of FGFR2 in its phosphorylated form, as well as HOXA9 and MEIS1 expression. In contrast, preincubation with the ATP-mimetic inhibitor PD173074, before FGF2 stimulation, reduced FGFR2 nuclear amount and HOXA9 and MEIS1 transcript level, thereby indicating that MLL-AF4 aberrant activity depends on the nuclear availability of FGFR2. Overall, our study identifies FGFR2 as a new and promising therapeutic target in t(4;11) leukemia.

Inhibition of 37/67kDa Laminin-1 Receptor Restores APP Maturation and Reduces Amyloid-ß in Human Skin Fibroblasts from Familial Alzheimer's Disease.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder caused by protein misfolding and aggregation, affecting brain function and causing dementia. Amyloid beta (Aß), a peptide deriving from amyloid precursor protein (APP) cleavage by-and γ-secretases, is considered a pathological hallmark of AD. Our previous study, together with several lines of evidence, identified a strict link between APP, Aß and 37/67kDa laminin receptor (LR), finding the possibility to regulate intracellular APP localization and maturation through modulation of the receptor. Here, we report that in fibroblasts from familial AD (fAD), APP was prevalently expressed as an immature isoform and accumulated preferentially in the transferrin-positive recycling compartment rather than in the Golgi apparatus. Moreover, besides the altered mitochondrial network exhibited by fAD patient cells, the levels of pAkt and pGSK3 were reduced in respect to healthy control fibroblasts and were accompanied by an increased amount of secreted Aß in conditioned medium from cell cultures. Interestingly, these features were reversed by inhibition of 37/67kDa LR by NSC47924 a small molecule that was able to rescue the "typical" APP localization in the Golgi apparatus, with consequences on the Aß level and mitochondrial network. Altogether, these findings suggest that 37/67kDa LR modulation may represent a useful tool to control APP trafficking and Aß levels with implications in Alzheimer's disease.

Liposome-Embedding Silicon Microparticle for Oxaliplatin Delivery in Tumor Chemotherapy.

Mesoporous silicon microparticles (MSMPs) can incorporate drug-carrying nanoparticles (NPs) into their pores. An NP-loaded MSMP is a multistage vector (MSV) that forms a Matryoshka-like structure that protects the therapeutic cargo from degradation and prevents its dilution in the circulation during delivery to tumor cells. We developed an MSV constituted by 1 µm discoidal MSMPs embedded with PEGylated liposomes containing oxaliplatin (oxa) which is a therapeutic agent for colorectal cancer (CRC). To obtain extra-small liposomes able to fit the 60 nm pores of MSMP, we tested several liposomal formulations, and identified two optimal compositions, with a prevalence of the rigid lipid 1,2-distearoyl-sn-glycero-3-phosphocholine and of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. To improve the MSV assembly, we optimized the liposome-loading inside the MSMP and achieved a five-fold increase of the payload using an innovative lyophilization approach. This procedure also increased the load and limited dimensional changes of the liposomes released from the MSV in vitro. Lastly, we found that the cytotoxic efficacy of oxa-loaded liposomes and-oxa-liposome-MSV in CRC cell culture was similar to that of free oxa. This study increases knowledge about extra-small liposomes and their loading into porous materials and provides useful hints about alternative strategies for designing drug-encapsulating NPs.

ZSCAN4+ mouse embryonic stem cells have an oxidative and flexible metabolic profile.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4+ mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4+ cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4+ stem cell state with potential applications in regenerative medicine.

New Insights into the Molecular Bases of Familial Alzheimer's Disease.

Like several neurodegenerative disorders, such as Prion and Parkinson diseases, Alzheimer's disease (AD) is characterized by spreading mechanism of aggregated proteins in the brain in a typical "prion-like" manner. Recent genetic studies have identified in four genes associated with inherited AD (amyloid precursor protein-APP, Presenilin-1, Presenilin-2 and Apolipoprotein E), rare mutations which cause dysregulation of APP processing and alterations of folding of the derived amyloid beta peptide (A). Accumulation and aggregation of A in the brain can trigger a series of intracellular events, including hyperphosphorylation of tau protein, leading to the pathological features of AD. However, mutations in these four genes account for a small of the total genetic risk for familial AD (FAD). Genome-wide association studies have recently led to the identification of additional AD candidate genes. Here, we review an update of well-established, highly penetrant FAD-causing genes with correlation to the protein misfolding pathway, and novel emerging candidate FAD genes, as well as inherited risk factors. Knowledge of these genes and of their correlated biochemical cascade will provide several potential targets for treatment of AD and aging-related disorders.

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aß) production and the amyloidogenic pathway, generating Aß, whose accumulation characterizes Alzheimer's disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aß and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aß, may mediate Aß-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3ß.

Microbiome Influence in the Pathogenesis of Prion and Alzheimer's Diseases.

Misfolded and abnormal ß-sheets forms of wild-type proteins, such as cellular prion protein (PrPC) and amyloid beta (Aß), are believed to be the vectors of neurodegenerative diseases, prion and Alzheimer's disease (AD), respectively. Increasing evidence highlights the "prion-like" seeding of protein aggregates as a mechanism for pathological spread in AD, tauopathy, as well as in other neurodegenerative diseases, such as Parkinson's. Mutations in both PrPC and Aß precursor protein (APP), have been associated with the pathogenesis of these fatal disorders with clear evidence for their pathogenic significance. In addition, a critical role for the gut microbiota is emerging; indeed, as a consequence of gut-brain axis alterations, the gut microbiota has been involved in the regulation of Aß production in AD and, through the microglial inflammation, in the amyloid fibril formation, in prion diseases. Here, we aim to review the role of microbiome ("the other human genome") alterations in AD and prion disease pathogenesis.

Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation.

PURPOSE: The t(4;11)(q21;q23) translocation characterizes a form of acute lymphoblastic leukemia with a poor prognosis. It results in a fusion gene encoding a chimeric transcription factor, MLL-AF4, that deregulates gene expression through a variety of still controversial mechanisms. To provide new insights into these mechanisms, we examined the interaction between AF4, the most common MLL fusion partner, and the scaffold protein 14-3-3θ, in the context of t(4;11)-positive leukemia. METHODS: Protein-protein interactions were analyzed using immunoprecipitation and in vitro binding assays, and by fluorescence microscopy in t(4;11)-positive RS4;11 and MV4-11 leukemia cells and in HEK293 cells. Protein and mRNA expression levels were determined by Western blotting and RT-qPCR, respectively. A 5-bromo-2'-deoxyuridine assay and an annexin V/propidium iodide assay were used to assess proliferation and apoptosis rates, respectively, in t(4;11)-positive and control cells. Chromatin immunoprecipitation was performed to assess binding of 14-3-3θ and AF4 to a specific promoter element. RESULTS: We found that AF4 and 14-3-3θ are nuclear interactors, that 14-3-3θ binds Ser588 of AF4 and that 14-3-3θ forms a complex with MLL-AF4. In addition, we found that in t(4;11)-positive cells, 14-3-3θ knockdown decreased the expression of MLL-AF4 target genes, induced apoptosis and hampered cell proliferation. Moreover, we found that 14-3-3θ knockdown impaired the recruitment of AF4, but not of MLL-AF4, to target chromatin. Overall, our data indicate that the activity of the chimeric transcription factor MLL-AF4 depends on the cellular availability of 14-3-3θ, which triggers the transactivating function and subsequent degradation of AF4. CONCLUSIONS: From our data we conclude that the scaffold protein 14-3-3θ enhances the aberrant activity of the chimeric transcription factor MLL-AF4 and, therefore, represents a new player in the molecular pathogenesis of t(4;11)-positive leukemia and a new promising therapeutic target.

HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-ß and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway. Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system.

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.

Attempt to Untangle the Prion-Like Misfolding Mechanism for Neurodegenerative Diseases.

The misfolding and aggregation of proteins is the neuropathological hallmark for numerous diseases including Alzheimer's disease, Parkinson's disease, and prion diseases. It is believed that misfolded and abnormal ß-sheets forms of wild-type proteins are the vectors of these diseases by acting as seeds for the aggregation of endogenous proteins. Cellular prion protein (PrPC) is a glycosyl-phosphatidyl-inositol (GPI) anchored glycoprotein that is able to misfold to a pathogenic isoform PrPSc, the causative agent of prion diseases which present as sporadic, dominantly inherited and transmissible infectious disorders. Increasing evidence highlights the importance of prion-like seeding as a mechanism for pathological spread in Alzheimer's disease and Tauopathy, as well as other neurodegenerative disorders. Here, we report the latest findings on the mechanisms controlling protein folding, focusing on the ER (Endoplasmic Reticulum) quality control of GPI-anchored proteins and describe the "prion-like" properties of amyloid-ß and tau assemblies. Furthermore, we highlight the importance of pathogenic assemblies interaction with protein and lipid membrane components and their implications in both prion and Alzheimer's diseases.

Protein Syndesmos is a novel RNA-binding protein that regulates primary cilia formation.

Syndesmos (SDOS) is a functionally poorly characterized protein that directly interacts with p53 binding protein 1 (53BP1) and regulates its recruitment to chromatin. We show here that SDOS interacts with another important cancer-linked protein, the chaperone TRAP1, associates with actively translating polyribosomes and represses translation. Moreover, we demonstrate that SDOS directly binds RNA in living cells. Combining individual gene expression profiling, nucleotide crosslinking and immunoprecipitation (iCLIP), and ribosome profiling, we discover several crucial pathways regulated post-transcriptionally by SDOS. Among them, we identify a small subset of mRNAs responsible for the biogenesis of primary cilium that have been linked to developmental and degenerative diseases, known as ciliopathies, and cancer. We discover that SDOS binds and regulates the translation of several of these mRNAs, controlling cilia development.