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Characterization of Soluble Protein Extracts from Keratinized Tissues: identification of Ubiquitin Universally Distributed in Hair, Nail, and Stratum Corneum
Abstract
Partial protein extracts were prepared from hair, nail, and stratum corneum in the absence of urea and interfacial surfactant. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoreses of these extracts showed low-molecular weight protein-rich patterns apparently different from those of whole protein extracts, which mainly consist of keratin bands. Several protein bands characterized each keratinized tissue or its derived species. In addition, we identified a major band of approximately 7 kDa as ubiquitin, a ubiquitously distributed protein that mediates non-lysosomal protein degradation, through direct amino acid sequence analysis of the electro-blotted protein band. The partial extraction is useful for investigation of soluble proteins retained in the keratinized tissues.
... We showed there was a detectable amount of nucDNA, mRNA and protein in the nail samples. Regarding the existence of other RNA or protein in nail samples, some studies have shown that xenobiotic RNA such as dermatophytes' mRNA exist in infected nails [35], and small internal proteins also exist in nails [7]. In this study, the amount of GAPDH mRNA and target protein varied widely among the three unrelated subjects. ...
... Nails are modified heavy keratin structures at the ends of the digits, contextures of which sensitively reflect the status of systemic blood supply [6]. Regarding the feasibility of accessing biomarkers from nails, not only protein [7-9] but also DNA [10-14] can be analyzed for molecular epidemiology research, medical diagnostics, and forensic science. It was possible to detect circulatory xenobiotic DNA such as hepatitis B virus in nail clippings among hepatitis B surface antigens positive patients [15]. ...
... Nails are modified heavy keratin structures at the ends of the digits, contextures of which sensitively reflect the status of systemic blood supply [6] . Regarding the feasibility of accessing biomarkers from nails, not only protein789 but also DNA1011121314 can be analyzed for molecular epidemiology research, medical diagnostics, and forensic science. It was possible to detect circulatory xenobiotic DNA such as hepatitis B virus in nail clippings among hepatitis B surface antigens positive pa- tients [15] . ...
Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples.
Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments.
The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study.
Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.
... Human skin consists of stratum corneum which is 70% keratin [2] while a human hair is 90% keratin [12,13]. The hair keratin consists of 50-60% alpha keratin while nail keratin, contrariwise, has characteristics of both hair and skin keratins in varying proportions [14,15], with 21.9% of amino acids being cysteine and serine [16]. Wool and feathers are the most abundant sources of keratin worldwide due to their utilisation in the textile and food industry, respectively [17,18]. ...
Keratin-based waste materials such as wool and waste chicken feathers are driving investigations to beneficiate them. The poultry industry in South Africa generates about 230 million kg of waste chicken feathers per annum, which makes them the abundant keratin source. most of which is disposed of by landfilling or combustion. The current disposal techniques through landfilling or combustion are not environmentally friendly. Thus, methods for beneficiation of the waste are needed. Considering that chicken feathers are comprised of mainly keratin, it is plausible that the keratin can be exploited for application in biomedical applications. However, keratin biomaterials have not found a breakthrough in clinical applications. The keratin can be recovered in the form of fibres or dissolved from feathers in suitable solvents. Regenerated keratin biomaterials can take the form of hydrogels, membranes, films, sponges, scaffolds, and nanofibres. These materials possess excellent properties that can be applied to different fields, including the health sector. Currently, there is no review paper that puts together all possible beneficiations of waste chicken feathers keratin in biomedicine. Therefore, this work exposes the chemistry and characteristics of keratin from different sources including chicken feather keratin in relation to their potential use in the biomedical applications. This review also highlights the properties of regenerated keratin and corresponding biomaterials, including electrospun regenerated keratin fibres for biomedical applications. Keratin nanofibres, also possess advanced properties for biomedical applications due to nanofibres reception in medical applications. Keratin is one biopolymer that can function as an acceptable biopolymer. The review indicates that there is a need for biopolymers as many fields rely on petroleum-based polymers which tend to have biocompatibility limits and are unsustainably resourced.
... However in case of diabetes mellitus, an increased concentration of glycated nail proteins is detected, explaining its usefulness as a diagnostic marker of this metabolic disorder of the carbohydrate metabolism [1,38]. Using SDS-PAGE, we identified in this study four bands in the nonglycated nail protein fraction and two bands in the glycated protein fraction, representing different epithelial keratins of the nail plate, with molecular masses ranging from 46 to 67 kDa [22,23,[39][40][41]. The epithelial keratins K1, K5, K6, K10, K14, K16 and K17 are expressed in the nail plate, whereas the nail bed expresses the suprabasal keratins (K6, K16 and K17) and the nail matrix the keratins K1, K5, K10 and K14 [26,42]. ...
Although assessment of glycated nail proteins may be a useful marker for monitoring of diabetes, their nature and formation are still poorly understood. Besides a detailed anatomical analysis of keratin glycation, the usefulness of glycated nail protein assessment for monitoring diabetic complications was investigated.
216 patients (94 males, 122 females; mean age ± standard deviation: 75.0 ± 8.7 years) were enrolled. Glycation of nail and eye lens proteins was assessed using a photometric nitroblue tetrazolium-based assay. Following chromatographic separation of extracted nail proteins, binding and nonbinding fractions were analyzed using one-dimensional gel electrophoresis. Using a hand piece containing a latch-type-bur, a meticulous cutting of the nail plate into superficial and deep layers was performed, followed by a differential analysis of fructosamine.
Using SDS PAGE, four and two bands were identified among the nonglycated and glycated nail fraction respectively. Significantly lower fructosamine concentrations were found in the superficial nail layer (mean: 2.16 ± 1.37 μmol/g nails) in comparison with the deep layer (mean: 4.36 ± 2.55 μmol/g nails) (P<0.05). A significant higher amount of glycated eye lens proteins was found in diabetes mellitus patients (mean: 3.80 ± 1.57 μmol/g eye lens) in comparison with nondiabetics (mean: 3.35 ± 1.34 μmol/g eye lens) (P<0.05). A marked correlation was found between glycated nail and glycated eye lens proteins [y (glycated nail proteins) = 0.39 + 0.99 x (eye lens glycated proteins); r2 = 0.58, P<0.001]. The concentration of glycated eye lens proteins and the HbA1c level were found to be predictors of the concentration of glycated nail proteins.
Glycation of nail proteins takes place in the deep layer of finger nails, which is in close contact with blood vessels and interstitial fluid. Glycation of nail proteins can be regarded as a representative marker for diabetic glycation-associated target organ damage.
... The bulb region was then incubated in buffer as described by Inoue et al. at 37 TC for 96 hr. 26 Following extraction, the proteins were dialyzed against 0.2 M Tris-HC1, pH 8.0 containing Complete Protease Inhibitor Cocktail tablets (Roche, Indianapolis, IN) at room temperature for 4 hr (2 buffer changes) and then at 4 TC overnight. Protein concentration was determined using the DC Protein Assay kit (BioRad, Hercules, CA). ...
Over the past fifty years, numerous studies have been performed to determine the health effects of exposure to organophosphorus (OP) nerve agents. The traditional approaches to determine OP exposure are invasive (e.g. require blood samples) laboratory-based assays that are not ideal for rapid, reliable testing in the event of a mass exposure scenario. However, advances in biomarker discovery make it possible to develop a less invasive, more expedient assay. The active hair follicle bulb expresses a large number of proteins, is easily accessible, and is an excellent candidate from which to assess biomarkers. In this report, we describe a novel immunohistochemistry (IHC) method developed for this initial hair follicle investigation, as well as the initial work completed to determine the presence of fifteen potential protein OP agent exposure biomarkers in the rat whisker follicle bulb. Each potential biomarker protein was chosen because of its change in expression in non-hair follicle tissue following exposure to pesticides and/or OP nerve agents. Using Western blotting, the expression of these proteins was verified in the rat whisker follicle bulb. Following verification in the digested hair follicle intact whole-hair IHC was performed to determine protein expression levels in the intact whisker follicle bulb.
... Keratins that are not polymerized in heterodimers are broken down (Kulesh et al. 1989). Keratins that need to be recycled in hepatocytes are marked for proteolysis in the proteasome by being tagged with ubiquitin (Inoue et al. 2001). In hepatocytes, the dephosphorylated K18 is broken down enzymatically by the proteases caspase-3 and -7 but the hyperphosphorylated forms of K18 cannot be cleaved by these caspases (Ku & Omary, 2001). ...
Historically, the term 'keratin' stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament-associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament-forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filament-forming proteins that were extracted from the living layers of the epidermis were grouped as 'prekeratins' or 'cytokeratins'. Currently, the term 'keratin' covers all intermediate filament-forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non-keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non-modified epidermis is a keratinized stratified epithelium, and that all other stratified and non-stratified epithelia are non-keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum. The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered.
Protein-based biomaterials have emerged as a promising alternative because of their inherent cell-to-cell interaction, structural support, and cellular communications. Over the last century, advances in the extraction, purification, and characterization of keratin proteins from wool, feathers, horns, and other animal sources have created a keratin-based biomaterials platform. Keratins, like many other naturally generated macromolecules, have biological activity and biocompatibility built-in. Furthermore, isolated keratins can self-assemble into structures that control cellular identification and behaviour. As a result, keratin biomaterials with applications in wound healing, drug delivery, tissue engineering, trauma, and medical devices have been developed due to these properties. This review examines the function of keratin in the human body in-depth, focusing on the history of keratin research and a current evaluation of emerging approaches in biomedical fields like tissue engineering, medical science, regenerative medicine, and drug delivery.
The nail is an important skin appendage, but not many dermatologists are aware of the importance it receives outside our specialty. This article focuses on the nail in non-dermatological contexts. The nail is a keratinized matrix capable of continuous growth with the ability to incorporate various compounds within its structure. Therefore it can be used to monitor long-term consumption of drugs. It is also an excellent source of germ-line DNA for genetic analyses. With an increased undrstanding of nail physiology, there is now a better understanding of its connection to various pathologies as well. Nails, being peripherally placed, are easy to sample without significant discomfort to the patient, making them a valuable diagnostic tool. For this narrative review, we carried out a PubMed search using the key words "nail clipping," "nail DNA," "nail diabetes mellitus;" "nail clipping oncology," and "nail forensics". Retrieved articles were searched for information pertaining to non-dermatologic uses of nail for evaluation, which is presented in a narrative fashion. It is clear from recent literature that the nail is not just an inert skin appendage, but a dynamic window into the ever-changing metabolic and genetic milieu. We highlight the numerous roles of nail specimens, as well as point towards future research needed therein.
We applied the Shindai method, which is an effective procedure for extracting proteins from human hair and wool, in the preparation of human nail protein solution. The amount of protein extracted by the Shindai method was about 2.5 times more than that obtained by the conventional method. Obtained proteins contained soft keratins in addition to hard keratins and matrix proteins. We found the presence of serine phosphorylation in keratins and matrix proteins, in addition to threonine and tyrosine phosphorylation in keratins, indicating the presence of post-translational modifications in the nail proteins. When we used pre-cast, post-cast, and soft post-cast methods that can convert hair protein solution into protein films, white color films were formed from the nail protein solution. The maximaum protein recovery from the films was 70-90%. The protein components mainly consisted of hard and soft keratins. Using the post-cast (pH 5) and soft postcast methods, the films formed slowly (approximately 5-40 min) and became translucent. In construct, the pre-cast and post-cast methods resulted in films which formed quickly (within 1 min) and turned white and opaque. SEM observations showed that the surfaces of the translucent films were smoother than the surfaces of the opaque films. Translucent nail protein films exhibited an absorption around 270-295 nm, indicating their selective application for UV-care goods.
Background: Various kinds of hair products are widely used due to the increase of interest in hair styling. Cosmetic procedures such as permanent waving are very popular today, but the medical studies related to the meaning and restoration pattern of hair damage are mainly based on structural findings. In measuring the degree of hair damage by the moleculobiological methods rather than the structural studies, the findings seem to be highly objective and standardized. Objective: The purpose of this study was to identify the patterns of hair damage and restoration through electrophoresis and western blot analysis of hair proteins. Methods: The three volunteers who we selected as subjects did not have any specific medical illness and had not performed any special cosmetic procedures which could have caused hair damage during the six months before the study. We conducted permanent waving on them. Human hair samples were obtained from the occipital scalp, which were that was not affected by the androgen. We performed extraction and concentration of the whole and partial hair protein, then operated electrophoresis and western blot analysis of the hair protein. Results: In the western blot analysis of whole hair proteins, there was one positive finding on subject A. This may have resulted from the small amount of partial proteins among the whole hair proteins. In the western blot analysis of partial hair proteins, subject A and B showed positive findings. In particular, positive findings were found on the 14th week of the experiment. Conclusion: These results show a change in the hair proteins due to hair damage, and ultrastructurally, we found the possibility of prologation of actual hair damage longer than expected.
Two types of peptide fractions were extracted from the human hairs and the properties for the fractions were investigated. The molecular weight of A fractions was 11 kDa, and that of B fractions 9.7 kDa. Artificially damaged human hairs were prepared by repeating the bleach treatments, and the recoveries in physical properties for the damaged hairs were estimated by impregnating those extracted peptide fractions. When the A fractions were impregnated into the damaged hairs, the formation of permanent waves was slightly recovered. As for the B fractions the tensile strengths for the damaged hairs were regained to some extent.
The time-dependent expression of ubiquitin (Ub) was examined in murine skin wounds and 55 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-5 days, III: 7-14 days and IV: 17-21 days). In murine skin wound specimens, neutrophils, macrophages and fibroblasts showed intensive Ub-positive reactions in the nuclei. In the human wound specimens with wound ages between 4 h and 1 day, neutrophils with strong intranuclear positive reactions for Ub were observed. With increasing wound ages, the nuclei of macrophages and fibroblasts were more intensively stained with anti-Ub antibody. Morphometrically, the intranuclear Ub-positive ratios were very low in group I. The skin wound specimens in groups II and IV showed Ub-positive ratios of >10%, and all samples in group III had Ub-positive ratios of >20%. These results suggest that a ratio of >10% for Ub indicates a wound age of at least 1 day. In contrast, Ub-positive ratios of less than 10% indicate a wound age of <1 day. Moreover, there was a significant difference in the Ub-positive ratio between group III and the other three groups. Thus, Ub-positive ratios considerably exceeding 30%, possibly indicate a wound age of 7-14 days. From the viewpoint of a forensic pathology application, the present study showed that Ub is suitable as a marker of wound age determination.
S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.
We previously found that certain hair proteins were soluble by means of a partial extraction method. In this study, we demonstrate that the amount of soluble proteins internally formed in permed and bleached hair, labile proteins, is a useful index for hair damage assessment. Compared to tensile property changes, this index rose in widely dynamic ranges as the time of either permanent waving or bleaching treatments increased. The amount of labile proteins was much larger than that of proteins eluted into perming and bleaching lotions. However, the labile proteins showed electrophoretic profiles similar to those of the eluted proteins. These results suggest that a portion of the stable proteins in normal hair was transformed into labile proteins upon permanent waving and bleaching treatments. Consequently, permed and bleached hair tends to release the resultant labile proteins.
Gephyrin is a bifunctional protein which is essential for both synaptic clustering of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (MoCo) in peripheral tissues. Mice deficient in gephyrin die early postnatally and display a loss of glycine receptors (GlyRs) and many GABA(A) receptor (GABA(A)R) subtypes from postsynaptic sites. In addition, the activities of the MoCo-dependent enzymes xanthine dehydrogenase and sulfite oxidase are reduced to background levels in the liver and intestine of these animals. To genetically separate the different consequences of gephyrin deficiency, we expressed a transgene of the plant homolog Cnx1, known to rescue mammalian MoCo deficiency, on the background of gephyrin knockout mice. Cnx1 partially restored sulfite oxidase activity in the liver of the transgenic animals, whereas early lethality and the loss of GlyR clustering were unaltered. Our data suggest that the loss of neurotransmitter receptor clustering at inhibitory synapses causes the early lethality of gephyrin deficient mice.
Because of small fluctuations, it is difficult to evaluate hair damage caused by bleaching using previously utilized hair damage indexes. Application of commercial bleaching products elevates partially extractable labile hair protein amounts in the range of 0.4-1.2 mg/g of hair. Within this range, the level of labile protein fluctuates greatly, depending on the extent of bleaching. In the current study, it was found that the effects of alkaline constituents and various peptides contained in bleaching lotions on hair damage could be evaluated by measuring labile protein amounts without employing harsher bleaching conditions.
Recent studies have shown that various tumor cells accumulate ubiquitin (Ub)-conjugated proteins, the profiles of which differ from those of normal cells. To identify the Ub-conjugated proteins accumulated specifically by human carcinoma cells, a two-dimensional immunoblot analysis of 31 surgically resected human primary colorectal carcinoma tissues was performed using an anti-Ub monoclonal antibody, KM691. Two distinct Mr 42,000 and 45,000 proteins in the Triton X-insoluble fractions of carcinoma tissues reacted with this antibody, whereas only one Mr 45,000 protein reacted in normal tissues. The Mr 42,000 Ub-conjugated proteins were specific to carcinoma tissues from 25 patients (80.6%). One of the purified Mr 42,000 proteins was digested with Achromobacter protease I. This protein was identified as a cytokeratin 8 (CK 8) fragment based on both molecular mass determination and molecular mass searching of Achromobacter protease I-digested fragments of proteins registered in a protein sequence data base. Two-dimensional immunoblot analysis with an anti-CK 8 antibody confirmed that all of the Mr 42,000 proteins were CK 8 degradation products. These results demonstrate that human colorectal carcinomas specifically accumulate Mr 42,000 Ub-conjugated CK 8 fragments. This accumulation was observed frequently not only in advanced (18/22, 81.8%), but also in early stage cases (7/9, 77.8%), suggesting that it occurs even in the early stages of colorectal carcinoma progression.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
In hair fiber, a cysteine-rich calcium-binding S100A3 protein is segregated in the inner part of the cuticle and postulated to play an important role in the attachment to the adjacent cuticular scale. In this study, elution of S100A3 from hair fiber was examined under various conditions by means of immunoblot analyses. The exposure of hair fiber to permanent waving lotions resulted in recoveries of substantial amounts of S100A3 by elution. Ultraviolet-light radiation and perming also increased the elution of S100A3 even without reductant. The distal part of hair fiber eluted less S100A3, as compared to the proximal section, under reducing conditions. These results suggest that S100A3 is eluted preferentially by daily washing and rinsing, especially from damaged hair. Given the presence of soluble S100A3 in the inner part of cuticle, we propose a new mechanism of hair damage in which the elution of S100A3 plays a major role.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
We investigated nail keratins obtained by sequential extraction with increasing reducing agent concentrations (50 mM and 200 mM 2-mercaptoethanol) and examined each of the extracted keratins by gel electrophoresis followed by immunoblotting. It was found that nail contained epidermal and hair keratins. With 50 mM 2-mercaptoethanol, only epidermal type of keratins including skin differentiation specific-keratin could be extracted, whereas hair type of keratins required 200 mM 2-mercaptoethanol for extraction from nail. These results may indicate that nail has unique properties with respect to it's keratin compositions. The difference in durabilities of epidermal and hair keratins in nail against 2-mercaptoethanol suggests that two different types of the keratin filaments which are composed of epidermal or hair keratin polypeptides may have differing structural features, despite of co-existence in nail.
A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.
The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event (Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., and Maier, C. (1990) Neuron 5, 411-419). Solid phase immunochemical assays measuring intersegmental muscle pools of free and conjugated ubiquitin reveal that the induction of polyubiquitin mRNA is accompanied by a proportional increase in total ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at eclosion, during which loss of muscle protein mass is maximum. A smaller but measurable increase in ubiquitin conjugates is observed earlier in pupal development coincident with a modest enhanced degradation of myofibrillar proteins. Accumulation of ubiquitin conjugates is accompanied by induction in the pathway for polypeptide ligation, including the activating enzyme (E1), several carrier protein (E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both accumulation of ubiquitin polypeptide and the enzymes of the conjugation pathway are subject to regulation by declining titers of the insect molting hormone 20-hydroxyecdysone, which signals onset of programmed cell death in the intersegmental muscles. Thus, programmed cell death within the intersegmental muscles is accomplished in part by stimulation of the ubiquitin-mediated degradative pathway through a coordinated induction of ubiquitin and the enzymes responsible for its conjugation to yield proteolytic intermediates. This suggests enzymes required for ubiquitin conjugation may represent additional genes recruited for developmentally programmed death.
Analyses on sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that the human hair cuticle extracts mainly consist of a 7-kDa component and keratin proteins. The S-carboxymethylation of the cuticle extracts made the 7-kDa band shift to the 15-kDa position. After electroblotting of the S-carboxymethyl derivative, the membrane pieces carrying the 15-kDa band were treated with trypsin and the released peptides were separated by reverse-phased HPLC. Amino acid sequence analyses revealed that the peptides corresponded to the partial sequences deduced from human genome coding for S100A3, a cysteine-rich calcium binding protein. The anti S100A3 serum, prepared by immunizing a synthetic peptide antigen, reacted with the 7-kDa and 15-kDa bands in immunoblotting analyses. Immunofluorescence microscopy showed intense labeling to the cuticular layer with the anti S100A3 serum. These results indicated that S100A3 was highly expressed in the human hair cuticle.
Keratins are the major structural proteins of the vertebrate epidermis and its appendages, constituting up to 85% of a fully differentiated keratinocyte. Together with actin microfilaments and microtubules, keratin filaments make up the cytoskeletons of vertebrate epithelial cells. Traced as far back in the evolutionary kingdom as mollusks, keratins belong to the superfamily of intermediate filament (IF) proteins that form alpha-helical coiled-coil dimers which associate laterally and end-to-end to form 10-nm diameter filaments. The evolutionary transition between organisms bearing an exoskeleton and those with an endoskeleton seemed to cause considerable change in keratin. Keratins expanded from a single gene to a multigene family. Of the approximately 60 IF genes in the human genome, half encode keratins, and at least 18 of these are expressed in skin. Vertebrate keratins are subdivided into two sequence types (I and II) that are typically coexpressed as specific pairs with complex expression patterns. The filament-forming capacity of a pair is dependent upon its intrinsic ability to self-assemble into coiled-coil heterodimers, a feature not required of the invertebrate keratins (Weber et al 1988). Approximately 20,000 heterodimers of type I and type II keratins assemble into an IF. Mutations that perturb keratin filament assembly in vitro can cause blistering human skin disorders in vivo. From studies of these diseases, an important function of keratins has been unraveled. These filaments impart mechanical strength to a keratinocyte, without which the cell becomes fragile and prone to rupturing upon physical stress. In this review, studies on the pattern of expression, structure, and function of skin keratins are summarized, and new insights into the functions of these proteins and their involvement in human disease are postulated.
The importance of wool in the textile industry has inspired extensive research into its structure since the 1960s. Over the past several years, however, the hair follicle has increased in significance as a system for studying developmental events and the process of terminal differentiation. The present chapter seeks to integrate the expanding literature and present a broad picture of what we know of the structure and formation of hair at the cellular and molecular level. We describe in detail the hair keratin proteins and their genes, their structure, function and regulation in the hair follicle, and also the major proteins and genes of the inner and outer root sheaths. We discuss hair follicle development with an emphasis on the factors involved and describe some hair genetic diseases and transgenic and gene knockout models because, in some cases, they stimulate natural mutations that are advancing our understanding of cellular interactions in the formation of hair.
Background:
In the translation of the genetic code each aminoacyl-tRNA synthetase (aaRS) must recognize its own (cognate) tRNA and attach the corresponding amino acid to the acceptor end of tRNA, discriminating all the others. The(alphabeta)2 phenylalanyl-tRNA synthetase (PheRS) is one of the most complex enzymes in the aaRS family and is characterized by anomalous charging properties. Structurally, the enzyme belongs to class II aaRSs, as its catalytic domain is built around an antiparallel beta sheet, but functionally it resembles class I as it aminoacylates the 2'OH of the terminal ribose of tRNA (class II aaRSs aminoacylate the 3'OH). With the availability of the three-dimensional structure of the complex between multisubunit PheRS and tRNAPhe, a fuller picture of the specific tRNA-aaRS interactions is beginning to emerge.
Results:
The crystal structure of Thermus thermophilus PheRS complexed with cognate tRNA has been solved at 3.28 A resolution. It reveals that one tRNAPhe molecule binds across all four PheRS subunits. The interactions of PheRS with tRNA stabilize the flexible N-terminal part of the alpha subunit, which appeared to form the enzyme's 11th domain, comprising a coiled-coil structure (helical arm) built up of two long antiparallel alpha helices. The helical arms are similar to those observed in SerRS and are in the same relative orientation with respect to the catalytic domain. Anticodon recognition upon tRNA binding is performed by the B8 domain, the structure of which is similar to that of the RNA-binding domain (RBD) of the small spliceosomal protein U1A. The Th. thermophilus PheRS approaches the anticodon loop from the minor groove side.
Conclusions:
The mode of interactions with tRNA explains the absolute necessity for the (alphabeta)2 architecture of PheRS. The interactions of tRNAPhe with PheRS and particularly with the coiled-coil domain of the alpha subunit result in conformational changes in TPsiC and D loops seen by comparison with uncomplexed yeast tRNAPhe. The tRNAPhe is a newly recognized type of RNA molecule specifically interacting with the RBD fold. In addition, a new type of anticodon-binding domain emerges in the aaRS family. The uniqueness of PheRS in charging 2'OH of tRNA is dictated by the size of its adenine-binding pocket and by the local conformation of the tRNA's CCA end.
Biochemical analysis indicated that the human nail plate contains two distinct types of keratin (skin-type and hair-type keratins), and several population of keratinocytes are thought to be associated with development of the nail. To elucidate the nature of the differentiation occurring in nail development, we examined the patterns of molecular markers relevant to the course of differentiation in the skin and hair in the nail matrix as well as in cultured nail cells. The nail matrix was characterized by the mutually exclusive localization of skin-type and hair-type markers, while in the apical matrix the localization of two groups of keratins partially overlapped. Double-label immunofluorescence showed the existence of unusual cells coexpressing both keratins, thereby indicating that the nail matrix consists of skin-type and hair-type and additionally intermediate-type differentiating cells. The cultured cells taken from the ventral matrix which develop under hair-type differentiation in vivo were found to express skin-type along with hair-type keratins, suggesting alteration of the pathway of differentiation in vitro. The cellular diversification as seen in in vitro cultured cells provides further insight into nail differentiation which is related to the multiple patterns of keratin expression that generate in the nail matrix.
To analyze the degradation system in epidermal cells during their generation, differentiation, and cell death, immunocytochemical localization of lysosomal cysteine and aspartic proteinases, an endogenous cysteine proteinase inhibitor, cystatin beta, and ubiquitin were examined using rat sole skin. By confocal laser microscopy, granular immunodeposits for lysosomal proteinases were well demonstrated in epidermal cells; immunoreactivity for cathepsins B and C was prominent in the lower spinous and basal layers, while that for cathepsins L and D was intense in the upper spinous and granular layers, although immunoreactivity for cathepsin D was also detected in the lower epidermal layers. Immunoreactivity for cathepsin H was weakly detected only in the spinous layer, where there were some intensely immunopositive cells with processes which were also immunopositive for S-100 alpha, indicating that these cells were Langerhans cells. Diffuse immunoreactivity for cystatin beta was intense in the spinous and granular layers and weak in the basal layer. In addition, we also examined the localization of ubiquitin, which is a signal peptide for cytosolic proteolysis; clear-cut granular immunodeposits for ubiquitin were detected in spinous and granular cells, and some were co-localized with cathepsin B immunoreactivity. In the basal layer, mitotic cells were strongly immunopositive for ubiquitin. These results suggest that cysteine and aspartic proteinases are involved in the lysosomal system of the epidermis, showing different distributions in the epidermal layers depending on the enzymes examined. Moreover, ubiquitin may be associated with the cell cycle-dependent degradation in basal cells while it also participates in the non-lysosomal proteolysis and probably, lysosomal proteolysis in the spinous and granular cells.
The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.
We have previously identified a cysteine-rich calcium binding protein S100A3 present in the cuticle of human hair fiber. In this study, we cloned a cDNA for mouse S100A3, identified its gene location, and elucidated the expression profile throughout hair follicle development. The mouse S100A3 gene was clustered with other S100 family members on chromosome 3, and specifically expressed in dorsal skin containing hair follicles. The level of S100A3 mRNA was elevated during the anagen phase of the hair growth cycle, and sharply declined from the regression phase on. In situ hybridization revealed that the S100A3 gene was prominently expressed in cuticular cells of the hair follicle, and mRNA levels were highest in the keratogenous zone over the entire cuticular layer. Expression was also observed to a lesser extent in differentiated cortical cells; however, expression was not observed in any other component of the hair follicle or dorsal tissues. Immunohistochemical analysis showed that the S100A3 protein accumulated in cuticular and cortical cells undergoing terminal differentiation. These results indicate that the S100A3 gene is exclusively expressed, and the translation product retained, in follicular cells differentiating into major components of the hair shaft. It seems likely that S100A3 plays an important role in calcium-dependent processes leading to hair shaft formation.
The structural proteins of hair: Isolation, characterization, and regulation of biosynthesis
- J M Gillespie