Alessia Nardi

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.

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.

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.

Transcriptional control of human steroid sulfatase

Steroid sulfatase (STS) is a membrane-bound microsomal enzyme that hydrolyzes various alkyl and aryl steroid sulfates, leading to the in situ formation of biologically active hormones. The entire human STS gene spans over approximately 200kbp of which the first 100kbp include the regulatory region, while the STS-coding region is located downstream. Previous studies indicated that STS expression, in different human tissues, could be regulated by at least six different promoters associated with alternative first exons. Here, we describe two new splicing patterns: the first, found in the prostatic cell line PC3, is based upon a partially coding new first exon (0d) that is spliced to a new second exon (1e). The second variant was found in the ovary and it is characterized by the novel splicing of the untranslated exon 0b to exon 0c, which is then spliced to the common exon 1b. We also report the results of a multiplex ligation-dependent probe amplification (RT-MLPA) analysis for the simultaneous detection, in qualitative and/or semi-quantitative terms, of the transcription patterns of STS in different tissues.

Isolation of a new class of cysteine-glycine-proline-rich beta-proteins (beta-keratins) and their expression in snake epidermis.

Scales of snakes contain hard proteins (beta‐keratins), now referred to as keratin‐associated beta‐proteins. In the present study we report the isolation, sequencing, and expression of a new group of these proteins from snake epidermis, designated cysteine–glycine–proline‐rich proteins. One deduced protein from expressed mRNAs contains 128 amino acids (12.5 kDa) with a theoretical pI at 7.95, containing 10.2% cysteine and 15.6% glycine. The sequences of two more snake cysteine–proline‐rich proteins have been identified from genomic DNA. In situ hybridization shows that the messengers for these proteins are present in the suprabasal and early differentiating beta‐cells of the renewing scale epidermis. The present study shows that snake scales, as previously seen in scales of lizards, contain cysteine‐rich beta‐proteins in addition to glycine‐rich beta‐proteins. These keratin‐associated beta‐proteins mix with intermediate filament keratins (alpha‐keratins) to produce the resistant corneous layer of snake scales. The specific proportion of these two subfamilies of proteins in different scales can determine various degrees of hardness in scales.

The expression of the human steroid sulfatase-encoding gene is driven by alternative first exons.

We have analyzed steroid sulfatase (STS) gene transcription in 10 human tissues: ovary, adrenal cortex, uterus, thyroid, liver, pancreas, colon, mammary gland, dermal papilla of the hair follicle, and peripheral mononuclear leukocytes. Overall, six different promoters were found to drive STS expression, giving rise to transcripts with unique first exons that were labeled 0a, 0b, 0c, 1a, 1c, and 1d, of which the last two and 0c are newly reported. All of them, except exon 1d, vary in length owing to the occurrence of multiple transcriptional start sites. While placental exon 1a is partially coding, the other five first exons are all untranslated. Three of these (0a, 0b, and 0c) are spliced to the common partially coding exon 1b, whereas the other two (1c and 1d) are spliced to the coding exon 2, which occurs in all transcripts. Whatever the ATG actually used, the differences are restricted to the signal peptide which is post-transcriptionally cleaved. Transcripts with exons 0a and 0b have the broadest tissue distribution, occurring, in 6 out of the 12 tissues so far investigated, while the other first exons are restricted to one or two tissues. The proximal promoter of each first exon was devoid of TATA box or initiator element and lacked consensus elements for transcription factors related to steroidogenesis, suggesting that regulatory sequences are probably placed at greater distance. In conclusion, the regulation of STS transcription appears to be more complex than previously thought, suggesting that this enzyme plays a substantial role in intercellular integration.

Identification of the 11 β-hydroxysteroid Dehydrogenase Type 1 mRNA and Protein in Human Mononuclear Leukocytes

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation.

RETRACTED: Occurrence of steroid-converting enzymes in the gonads of the Manila clam, Ruditapes philippinarum (Adams and Reeve, 1850), and correlation with the gametogenic cycle.

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