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Biomedical subjects

Stefano Biffo

Publications and source records attributed to Stefano Biffo.

12 recordsLinked to original sources
PubMed2026

KDM6A loss enhances oxidative phosphorylation uncovering tissue-level convergent evolution.

The tumor suppressor KDM6A/UTX, a histone demethylase and a 2-oxoglutarate-dependent dioxygenase, is frequently lost in many cancer types. We show that KDM6A loss pervasively activates oxidative phosphorylation in several solid tumors, generating a pseudo-hyperoxic environment, opposite from the pseudo-hypoxia observed in VHL-mutated renal carcinomas. Mechanistically, KDM6A sustains the expression of the coil-coil domain gene CCDC3, which inhibits CREB1-driven transcription of the mitochondrial regulator PPARGC1A. In the hematological cancer multiple myeloma where KDM6A is frequently deleted, its loss similarly promotes oxidative phosphorylation, but via an alternative mechanism: the increased transfer of mitochondria from stromal to myeloma cells via tunneling nanotubes, triggered by the loss of the mTORC1 inhibitor TRAF3IP3. Beyond cancer, KDM6A regulates oxidative phosphorylation also during development and in adult tissues, engaging either the CCDC3-CREB1 or the TRAF3IP3-mTORC1 pathways. These mutually exclusive associations suggest a tissue-level convergent evolution, positioning KDM6A as a central modulator of mitochondrial activity through context-specific partners.

Journal Article↗
PubMed2006

Nuclear myosin VI enhances RNA polymerase II-dependent transcription.

Myosin VI is the only myosin that moves toward the minus end of actin filaments, suggesting a unique biological function. Here, we show that myosin VI is present in the nucleus of mammalian cells where it colocalizes with newly transcribed mRNA and with RNA polymerase II (RNAPII) and is detected in the RNAPII complex. The colocalization and interaction of myosin VI with RNAPII require transcriptional activity. Chromatin immunoprecipitation (ChIP) demonstrates that myosin VI is recruited to the promoter and intragenic regions of active genes, encoding urokinase plasminogen activator (uPA), eukaryotic initiation factor 6 (p27/eIF6), and low-density lipoprotein receptor (LDLR), but not to noncoding, nonregulatory intergenic regions. Downregulation of myosin VI reduces steady-state mRNA levels of these genes in vivo, and antibodies to myosin VI reduce transcription in vitro. We suggest that myosin VI modulates RNAPII-dependent transcription of active genes, implicating the possibility of an actin-myosin based mechanism of transcription.

Cell Nucleus↗
PubMed2006

GABP complex regulates transcription of eIF6 (p27BBP), an essential trans-acting factor in ribosome biogenesis.

Eukaryotic initiation factor 6 (eIF6, alias p27BBP) is required for the biogenesis of 60S ribosomal subunits. eIF6 expression levels are tightly regulated in vivo, where they correlate with cellular growth. We analyzed how transcriptional regulation of eIF6 is achieved. We show that the human eIF6 promoter contains consensus sites for the GABP (GA-binding protein) transcription factor complex. Functional analysis of GABP consensus sequences by point mutations, EMSA (electrophoretic mobility shift assay) and a dominant negative mutant indicates that GABP is essential for eIF6 promoter activity. These data strengthen the hypothesis that GABP is a global regulator of ribosome synthesis.

Carrier Proteins↗
PubMed2005

Ferroportin is a monomer in vivo in mice.

Ferroportin (FPN) is the main iron export protein in mammals. The actual structure of FPN in vivo and the pathogenesis of ferroportin-related disease are unknown. We aimed at studying the structure and biochemical properties of FPN in mouse tissues that are key for iron homeostasis during various iron manipulations in vivo. We performed glycosylation and oligomerization studies in spleen and liver extracts from mice fed a standard, iron-deprived or iron-enriched diet for 5 months. Purification by affinity chromatography and sucrose gradient show that FPN is not part of a large multiprotein complex. Dietary manipulations did not affect the monomeric status of the native or denatured protein. The glycosylation studies showed that ferroportin is digested by peptide: N-glycosidase F but not by endoglycosidase H. The same results were obtained using protein extracts from iron-deficient or iron-loaded mice. In conclusion, our studies indicate that mouse FPN, regardless of the tissue iron status, is glycosylated but not enriched in mannose residues, and that exists mainly in monomeric form. The latter finding may have important implications for understanding the pathogenesis of the disease due to ferroportin mutations.

Animals↗
PubMed2005

Sen34p depletion blocks tRNA splicing in vivo and delays rRNA processing.

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.

Carrier Proteins↗
PubMed2005

Fibronectin controls cap-dependent translation through beta1 integrin and eukaryotic initiation factors 4 and 2 coordinated pathways.

Fibronectin (FN) is a major matrix protein involved in multiple processes. Little is known about how adhesion to FN affects the translational machinery. We show that in fibroblasts adhesion to FN triggers translation through the coordinated regulation of eukaryotic initiation factors (eIFs) 4F and 2 and is impaired by blocking beta1 integrin engagement. FN-stimulated translation has unique properties: (i) it is highly sensitive to the inhibition of phosphatidylinositol 3-kinase (PI3K), but not to the inhibition of mammalian target of rapamycin, downstream of PI3K; (ii) there is no synergy between serum-stimulated translation and FN-dependent translation; (iii) FN-dependent translation, unlike growth factor-stimulated translation, does not lead to increased translocation of 5' terminal oligopyrimidine tract mRNAs to polysomes; and (iv) cells devoid of attachment to matrix show an impairment of initiation of translation accompanied by phosphorylation of eIF2alpha, which cannot be reverted by active PI3K. These findings indicate that integrins may recruit the translational machinery in a unique way and that FN-dependent translation cannot be blocked by mammalian target of rapamycin inhibition.

Animals↗
PubMed2004

Overexpression of p27BBP in head and neck carcinomas and their lymph node metastases.

BACKGROUND: p27(BBP) is a regulator of ribosome assembly and an essential nuclear and cytoplasmic component of eukaryotes. METHODS: We investigated the immunochemical distribution of p27(BBP) in head and neck carcinomas, in the associated normal mucosa, and in regional lymph nodes. RESULTS: p27(BBP) is detectable in mucosal cells but is overexpressed in carcinomas, highly concentrated in large polymorphous nucleoli, and even larger and more evident in lymph node metastatic foci. Western blotting confirms increased p27(BBP) in carcinomas versus normal mucosa and also in metastatic versus normal lymph nodes. The overexpression of p27(BBP) corresponds to mRNA upregulation in carcinomas. Unexpectedly, a 52-kDa band specifically reacting with antibodies to p27(BBP) was observed in several carcinomas. CONCLUSIONS: p27(BBP) alterations are common events in the transition to malignancy and are probably involved in squamous carcinoma progression. Immune reagents raised to p27(BBP) may provide additional diagnostic tools for surgical pathology of tumor boundaries and lymph nodes. The 52-kDa band may represent an abnormal form of p27(BBP) expressed by transformed airway epithelia.

Adult↗
PubMed2003

Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly.

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.

Animals↗
PubMed2002

Formation of nuclear matrix filaments by p27(BBP)/eIF6.

p27(BBP)/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of beta 4 integrin. In cultured cells, a conspicuous fraction of p27(BBP)/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27(BBP)/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27(BBP)/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27(BBP)/eIF6 binds beta 4 integrin. Upon dialysis against IF buffer, p27(BBP)/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27(BBP)/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.

Animals↗
PubMed2002

BDNF binding to truncated trkB.T1 does not affect gene expression.

Truncated trkB.T1 is a splice variant of the neurotrophin receptor trkB. In spite of its abundance, and ability to bind and internalize BDNF, it is not clear whether it can transmit BDNF signaling. We tested this hypothesis by searching for proteins binding the evolutionarily conserved cyto-domain of trkB.T1, and by studying BDNF-induced changes of gene expression through DNA microarrays. Cells bearing trkB.T1 receptors presented morphological changes. However, no cytoplasmic interactors of trkB.T1 were found. In addition, BDNF-dependent modulation of gene expression was detected in cells bearing trkB.TK but not trkB.T1 receptors. These results suggest that the main function of trkB.T1 is to regulate local availability of neurotrophins and that it is unable to sense changes in BDNF availability.

3T3 Cells↗
PubMed1992

Bulbectomy Enhances Neurogenesis and Cell Turnover of Primary Olfactory Neurons But Does Not Abolish Carnosine Expression.

Primary olfactory neurons located in the olfactory neuroepithelium project to the ipsilateral olfactory bulb and undergo a continuous process of neurogenesis and differentiation. We describe, in the adult rat, the kinetics of proliferation, differentiation and survival of primary olfactory neurons either in the presence or absence of their target, the olfactory bulb. The experimental design included unilateral bulbectomy, coupled with a single bromodeoxyuridine pulse 35 days after surgery. The rate of proliferation and survival of olfactory neurons was then examined by immunohistochemistry for bromodeoxyuridine, and the differentiation status by in situ hybridization for calmodulin messenger RNA in immature and mature olfactory neurons and immunohistochemistry for the dipeptide carnosine in mature olfactory neurons. We show that primary olfactory neurons can synthesize carnosine in the absence of the olfactory bulb. However, the number of carnosine-immunopositive neurons in the absence of their target is dramatically reduced to less than one-fourth, whereas the number of olfactory neurons expressing calmodulin messenger RNA is only slightly reduced. The numeric reduction of carnosine-positive neurons in the target-deprived neuroepithelium is correlated with a dramatic reduction in the survival rate of olfactory neurons, since newly generated olfactory neurons are completely lost 35 days after the bromodeoxyuridine pulse. In contrast, in the normal olfactory neuroepithelium almost one-third of newly generated olfactory neurons survive 35 days after the bromodeoxyuridine pulse. On the whole, these data indicate that most of the primary olfactory neurons have a short lifespan but that once they have connected with the olfactory bulb they may persist longer, and suggest that throughout adulthood olfactory neurons are overproduced, differentiate independently from their target, and then undergo a process of target-induced neuronal selection.

Journal Article↗