Joseph A. Rothnagel

Identification of a major keratinocyte cell envelope protein, loricrin

During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. Transcripts of this gene are restricted to the granular layer and are as abundant as the differentiation-specific keratins, K1 and K10. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin.

Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is a hereditary skin disorder characterized by blistering and a marked thickening of the stratum corneum. In one family, affected individuals exhibited a mutation in the highly conserved carboxyl terminal of the rod domain of keratin 1. In two other families, affected individuals had mutations in the highly conserved amino terminal of the rod domain of keratin 10. Structural analysis of these mutations predicts that heterodimer formation would be unaffected, although filament assembly and elongation would be severely compromised. These data imply that an intact keratin intermediate filament network is required for the maintenance of both cellular and tissue integrity.

Emerging evidence for functional peptides encoded by short open reading frames

Short open reading frames (sORFs) are a common feature of all genomes, but their coding potential has mostly been disregarded, partly because of the difficulty in determining whether these sequences are translated. Recent innovations in computing, proteomics and high-throughput analyses of translation start sites have begun to address this challenge and have identified hundreds of putative coding sORFs. The translation of some of these has been confirmed, although the contribution of their peptide products to cellular functions remains largely unknown. This Review examines this hitherto overlooked component of the proteome and considers potential roles for sORF-encoded peptides.

Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation

Overexpression of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 has been observed in a variety of tumour types, however, it is unknown whether this dysregulation is a consequence of, or a driving force for, unregulated cell proliferation. We have shown that the levels of hnRNPs A1, A2 and B1, but not A3, are modulated during the cell cycle of Colo16 squamous carcinoma cells and HaCaT immortalized keratinocytes, suggesting that A1, A2 and B1 are needed at particular cell cycle stages. However, the levels of hnRNP A1, A2 and B1 mRNAs were constant, indicating that regulation of protein levels was controlled at the level of translation. RNAi suppression of hnRNP A1 or A3 alone did not affect the proliferation of Colo16 cells but the proliferation rate was significantly reduced when both were suppressed simultaneously, or when either was suppressed together with hnRNP A2. Reducing hnRNP A2 expression in Colo16 and HaCaT cells by RNAi led to a non-apoptotic-related decrease in cell proliferation, reinforcing the view that this protein is required for cell proliferation. Suppression of hnRNP A2 in Colo16 cells was associated with increased p21 levels but p53 levels remained unchanged. In addition, expression of BRCA1 was downregulated, at both mRNA and protein levels. The observed effects of hnRNP A2 and its isoforms on cell proliferation and their correlation with BRCA1 and p21 expression suggest that these hnRNP proteins play a role in cell proliferation.

Enzymatic recognition of 2'-modified ribonucleoside 5'-triphosphates: towards the evolution of versatile aptamers.

The quest for effective, selective and nontoxic nucleic‐acid‐based drugs has led to designing modifications of naturally occurring nucleosides. A number of modified nucleic acids have been made in the past decades in the hope that they would prove useful in target‐validation studies and therapeutic applications involving antisense, RNAi, aptamer, and ribozyme‐based technologies. Since their invention in the early 1990s, aptamers have emerged as a very promising class of therapeutics, with one drug entering the market for the treatment of age‐related macular degeneration. To combat the limitations of aptamers containing naturally occurring nucleotides, chemically modified nucleotides have to be used. In order to apply modified nucleotides in aptamer drug development, their enzyme‐recognition capabilities must be understood. For this purpose, several modified nucleoside 5′‐triphosphates were synthesized and investigated as substrates for various enzymes. Herein, we review studies on the enzyme‐recognition of various 2′‐sugar‐modified NTPs that were carried out with a view to their effective utilization in SELEX processes to generate versatile aptamers.

Overexpression of sonic hedgehog suppresses embryonic hair follicle morphogenesis

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development, we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype.

Differential subcellular distributions and trafficking functions of hnRNP A2/B1 spliceoforms.

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.

Keratin Bundling Proteins

Publisher Summary Keratin bundling proteins are a subcategory of intermediate filament associated proteins (IFAPs) that are defined by their ability to organize keratin filaments into macrofibrilar arrays; however, only filaggrin and a related protein have been shown unequivocally to bundle keratin and other intermediate filaments into parallel ropelike structures. This chapter discusses the keratin bundling proteins (particularly the filaggrin—the archetypal keratin bundling protein), filament bundling assays, techniques for the isolation of keratin intermediate filaments, and macrofibril formation assay. Only two proteins—filaggrin and filaggrin-2—have been shown to function as keratin-bundling proteins. It remains to be seen whether this exclusive group will acquire new members, with trichohyalin, hornerin, and repetin all requiring further testing. Although, this chapter focuses on keratin-bundling proteins, it is probable that novel or known IFAPs will be identified that bundle other intermediate filament types such as vimentin and desmin. In addition, the remarkably linear arrays of neurofilaments observed in some axons may be mediated by a specific bundling protein, and the role of neural IFAPs in this activity would seem worthy of further investigation.

Transglutaminase-mediated cross-linking in mammalian epidermis

SummaryThe ε-(γ-glutamyl)lysine isopeptide bond has been identified in certain structural proteins of the hair fibre and the epidermis. The major cross-linked proteins are not keratins and generally have little or no cysteine, but have a high glutamic acid/ glutamine residue content. In the hair fibre the cross-link appears in the citrulline-containing proteins of the medulla and the inner root sheath of the follicle. In the epidermis the cross-linked proteins are involved in the formation of the cornified envelope of the stratum corneum cells. In both cases, the cross-linked proteins contribute the characteristic property of chemical resistance to their b biological structures.

Citrulline in proteins from the enzymatic deimination of arginine residues.

Publisher Summary The occurrence of citrulline is restricted to specialized proteins whose tissues of origin contain the requisite enzyme for the conversion of the guanido side chain of certain arginine residues to the ureido side chains of citrulline. The tissues of importance are primarily vertebrate epidermis and the derived structures of it, although it has been reported that citrulline is present in a protein of nerve myelin. This chapter discusses the identification and estimation of citrulline in proteins. Citrulline gives a yellow color when allowed to react with Erhlich reagent, p -dimethylaminobenzaldehyde (1% w/v) in 0.5-1.0 M HCI. Citrulline can be estimated in acid hydrolysates of proteins and peptides using a standard amino acid analyzer. However, to elute the citrulline as a separate peak, it is necessary to alter the conditions of elution. The colorimetric determination of citrulline in proteins and peptides utilizes the reaction of ureido groups with diacetyl monoxime. Difficulties can be experienced with the quantitation of citrulline when colorimetric methods are applied directly to proteins. These difficulties include nonspecific color reactions and the appearance of trace amounts of suspended material in the reaction mixture. Consequently, it is advantageous to precede the colorimetric reaction by acid hydrolysis of the proteins.