Luisa Dalla Valle

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

Identification of reptilian genes encoding hair keratin-like proteins suggests a new scenario for the evolutionary origin of hair

The appearance of hair is one of the main evolutionary innovations in the amniote lineage leading to mammals. The main components of mammalian hair are cysteine-rich type I and type II keratins, also known as hard α-keratins or “hair keratins.” To determine the evolutionary history of these important structural proteins, we compared the genomic loci of the human hair keratin genes with the homologous loci of the chicken and of the green anole lizard Anolis carolinenis. The genome of the chicken contained one type II hair keratin-like gene, and the lizard genome contained two type I and four type II hair keratin-like genes. Orthology of the latter genes and mammalian hair keratins was supported by gene locus synteny, conserved exon–intron organization, and amino acid sequence similarity of the encoded proteins. The lizard hair keratin-like genes were expressed most strongly in the digits, indicating a role in claw formation. In addition, we identified a novel group of reptilian cysteine-rich type I keratins that lack homologues in mammals. Our data show that cysteine-rich α-keratins are not restricted to mammals and suggest that the evolution of mammalian hair involved the co-option of pre-existing structural proteins.

Evolution of hard proteins in the sauropsid integument in relation to the cornification of skin derivatives in amniotes

Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendages probably derived from the localization of specialized areas of dermal–epidermal interaction in the integument. The horny scales and the other derivatives were formed from large areas of dermal–epidermal interaction. The evolution of these skin appendages was characterized by the production of specific coiled‐coil keratins and associated proteins in the inter‐filament matrix. Unlike mammalian keratin‐associated proteins, those of sauropsids contain a double beta‐folded sequence of about 20 amino acids, known as the core‐box. The core‐box shows 60%–95% sequence identity with known reptilian and avian proteins. The core‐box determines the polymerization of these proteins into filaments indicated as beta‐keratin filaments. The nucleotide and derived amino acid sequences for these sauropsid keratin‐associated proteins are presented in conjunction with a hypothesis about their evolution in reptiles‐birds compared to mammalian keratin‐associated proteins. It is suggested that genes coding for ancestral glycine‐serine‐rich sequences of alpha‐keratins produced a new class of small matrix proteins. In sauropsids, matrix proteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestral protein. This mutation gave rise to the core‐box, and other regions of the original protein evolved differently in the various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteine proline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the high glycine proline tyrosine, non‐feather proteins, and into the glycine‐tyrosine‐poor group of feather proteins, which evolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. In therapsids‐mammals, mutations of the ancestral proteins formed the high glycine‐tyrosine or the high cysteine proteins but no core‐box was produced in the matrix proteins of the hard corneous material of mammalian derivatives.

Isolation of a mRNA encoding a glycine‐proline–rich β‐keratin expressed in the regenerating epidermis of lizard

During scale regeneration in lizard tail, an active differentiation of β‐keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard β‐keratin has been obtained from mRNA isolated from regenerating epidermis. Degenerate oligonucleotides, selected from the translated amino acid sequence of a lizard claw protein, were used to amplify a specific lizard keratin cDNA fragment from the mRNA after reverse transcription with poly dT primer and subsequent polymerase chain reaction (3′‐rapid amplification of cDNA ends analysis, 3′‐RACE). The new sequence was used to design specific primers to obtain the complete cDNA sequence by 5′‐RACE. The 835‐nucleotide cDNA sequence encodes a glycine‐proline–rich protein containing 163 amino acids with a molecular mass of 15.5 kDa; 4.3% of its amino acids is represented by cysteine, 4.9% by tyrosine, 8.0% by proline, and 29.4% by glycine. Tyrosine is linked to glycine, and proline is present mainly in the central region of the protein. Repeated glycine–glycine‐X and glycine‐X amino acid sequences are localized near the N‐amino and C‐terminal regions. The protein has the central amino acid region similar to that of claw–feather, whereas the head and tail regions are similar to glycine‐tyrosine–rich proteins of mammalian hairs. In situ hybridization analysis at light and electron microscope reveals that the corresponding mRNA is expressed in cells of the differentiating β‐layers of the regenerating scales. The synthesis of β‐keratin from its mRNA occurs among ribosomes or is associated with the surface of β‐keratin filaments. Developmental Dynamics 234:934–947, 2005.

Cloning and characterization of scale β-keratins in the differentiating epidermis of geckoes show they are glycine-proline-serine–rich proteins with a central motif homologous to avian β-keratins

The β‐keratins constitute the hard epidermis and adhesive setae of gecko lizards. Nucleotide and amino acid sequences of β‐keratins in epidermis of gecko lizards were cloned from mRNAs. Specific oligonucleotides were used to amplify by 3′‐ and 5′‐rapid amplification of cDNA ends analyses five specific gecko β‐keratin cDNA sequences. The cDNA coding sequences encoded putative glycine‐proline‐serine–rich proteins of 16.8–18 kDa containing 169–191 amino acids, especially 17.8–23% glycine, 8.4–14.8% proline, 14.2–18.1% serine. Glycine‐rich repeats are localized toward the initial and end regions of the protein, while a central region, rich in proline, has a strand conformation (β‐pleated fold) likely responsible for the formation of β‐keratin filaments. It shows high homology with a core region of other lizard keratins, avian scale, and feather keratins. Northern blotting and reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis show a higher β‐keratin gene expression in regenerating epidermis compared with normal epidermis. In situ hybridization confirms that mRNAs for these proteins are expressed in cells of the differentiating oberhautchen cells and β‐cells. Expression in adhesive setae of climbing lamellae was shown by RT‐PCR. Southern blotting analysis revealed that the proteins are encoded by a multigene family. PCR analysis showed that the genes are presumably located in tandem along the DNA and are transcribed from the same DNA strand like in avian β‐keratins. Developmental Dynamics 236:374–388, 2007.

Developmentally Regulated Expression and Activity of 17α-Hydroxylase/C-17,20-Lyase Cytochrome P450 in Rat Liver1

We have investigated the developmental pattern of expression and activity of 17α-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein·min) at a level comparable to that in the testis (188 pmol/mg protein·min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein·min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein·min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 ex...

Beta-keratins of turtle shell are glycine-proline-tyrosine rich proteins similar to those of crocodilians and birds

This study presents, for the first time, sequences of five beta‐keratin cDNAs from turtle epidermis obtained by means of 5′‐ and 3′‐rapid amplification of cDNA ends (RACE) analyses. The deduced amino acid sequences correspond to distinct glycine‐proline‐serine‐tyrosine rich proteins containing 122–174 amino acids. In situ hybridization shows that beta‐keratin mRNAs are expressed in cells of the differentiating beta‐layers of the shell scutes. Southern blotting analysis reveals that turtle beta‐keratins belong to a well‐conserved multigene family. This result was confirmed by the amplification and sequencing of 13 genomic fragments corresponding to beta‐keratin genes. Like snake, crocodile and avian beta‐keratin genes, turtle beta‐keratins contain an intron that interrupts the 5′‐untranslated region. The length of the intron is variable, ranging from 0.35 to 1.00 kb. One of the sequences obtained from genomic amplifications corresponds to one of the five sequences obtained from cDNA cloning; thus, sequences of a total of 17 turtle beta‐keratins were determined in the present study. The predicted molecular weight of the 17 different deduced proteins range from 11.9 to 17.0 kDa with a predicted isoelectric point of 6.8–8.4; therefore, they are neutral to basic proteins. A central region rich in proline and with beta‐strand conformation shows high conservation with other reptilian and avian beta‐keratins, and it is likely involved in their polymerization. Glycine repeat regions, often containing tyrosine, are localized toward the C‐terminus. Phylogenetic analysis shows that turtle beta‐keratins are more similar to crocodilian and avian beta‐keratins than to those of lizards and snakes.

BPA-Induced Deregulation Of Epigenetic Patterns: Effects On Female Zebrafish Reproduction

Bisphenol A (BPA) is one of the commonest Endocrine Disruptor Compounds worldwide. It interferes with vertebrate reproduction, possibly by inducing deregulation of epigenetic mechanisms. To determine its effects on female reproductive physiology and investigate whether changes in the expression levels of genes related to reproduction are caused by histone modifications, BPA concentrations consistent with environmental exposure were administered to zebrafish for three weeks. Effects on oocyte growth and maturation, autophagy and apoptosis processes, histone modifications, and DNA methylation were assessed by Real-Time PCR (qPCR), histology, and chromatin immunoprecipitation combined with qPCR analysis (ChIP-qPCR). The results showed that 5 μg/L BPA down-regulated oocyte maturation-promoting signals, likely through changes in the chromatin structure mediated by histone modifications, and promoted apoptosis in mature follicles. These data indicate that the negative effects of BPA on the female reproductive system may be due to its upstream ability to deregulate epigenetic mechanism.

Hard cornification in reptilian epidermis in comparison to cornification in mammalian epidermis

Abstract:  The structure of reptilian hard (beta)‐keratins, their nucleotide and amino acid sequence, and the organization of their genes are presented. These 13–19 kDa proteins are basic, rich in glycine, proline and serine, and different from cytokeratins. Their mRNAs are expressed in beta‐cells. The central part of beta‐keratins (this region has been previously termed ‘core‐box’ and is peculiar of all sauropsid proteins) is composed of two beta‐folded regions and shows a high identity with avian beta‐keratins. This central part present in all beta‐keratins, including feather keratins, is the site of polymerization to build the framework of beta‐keratin filaments. Beta‐keratins appear cytokeratin‐associated proteins. Their central region might have originated in an ancestral glycine‐rich protein present in stem reptiles from which beta‐keratins evolved and diversified into reptiles and birds. Stem reptiles of the Carboniferous period might have possessed glycine‐rich proteins derived from exons/domains corresponding to the variable, glycine‐rich region of cytokeratins. Beta‐keratins might have derived from a gene coding for small glycine‐rich keratin‐associated proteins. The glycine‐rich regions evolved differently in the lineage leading to modern reptiles and birds versus that leading to mammals. In the reptilian lineage some amino acid regions produced by point mutations and amino acid changes might have given rise to originate the central beta‐pleated region. The latter allowed the formation of filamentous proteins (beta‐keratins) associated with intermediate filament keratins and replaced them in beta‐keratin cells. In the mammalian lineage no beta‐pleated region was generated in their matrix proteins, the glycine‐rich keratin‐associated proteins. The latter evolved as glycine‐tyrosine‐rich, sulphur‐rich, and ultra‐sulphur‐rich proteins that are used for building hairs, horns and nails.

Analysis of Gene Expression in Gecko Digital Adhesive Pads Indicates Significant Production of Cysteine-and Glycine-Rich Beta-Keratins

Microscopic bristles (setae) present on digital pads permit the adhesion and climbing of geckos. Keratins of setae of the lizard Gekko gecko (Tokay gecko) were analyzed by the isolation of expressed mRNAs and by the generation of an EST library. Of the 510 sequences determined, 268 (52.9%) were unique. Of these, 14 appeared to encode alpha- and 111 beta-keratins. Within the beta-keratins, we identified five groups based on nucleotide sequence comparisons. Of these, one contained the bulk of beta-keratins, with 103 EST members. The mRNAs within this major group, together with two singlets, encoded cysteine-proline-serine-rich proteins of 10-14 kDa (Ge-cprp). One of the smaller groups of transcripts encoded slightly larger glycine-proline-serine-rich proteins, of 14-19 kDa (Ge-gprp). The remaining group consisted of smaller (9 kDa) serine-tyrosine-rich beta-keratins (Ge-strp). Thus three classes could be distinguished by amino acid sequence alignment. Exact matches for some of the peptide sequences obtained from setal proteins by ms/ms sequencing occur within several of these clones. Most of the beta-keratins were basic and contained a core-box region of two beta-strand sequences, with high homology to core-boxes present in avian scale and feather beta-keratins. Core-boxes are beta-folded regions that are likely responsible for polymerization into the beta-keratin filaments. The two deduced alpha-keratins of 52.7 kDa are both acidic, and contain the typical central rod region with some homology to mammalian and avian alpha-keratins, with variable N- and C-terminal regions. Basic beta-keratins and acidic alpha-keratins may interact electrostatically to form the resistant corneous material of setae.