Epidermal Protein Synthesis Research Articles

Transcriptomic and Proteomic Analyses of Myzus persicae Carrying Brassica Yellows Virus.

Viruses in the genus Polerovirus infect a wide range of crop plants and cause severe economic crop losses. BrYV belongs to the genus Polerovirus and is transmitted by Myzus persicae. However, the changes in transcriptome and proteome profiles of M. persicae during viral infection are unclear. Here, RNA-Seq and TMT-based quantitative proteomic analysis were performed to compare the differences between viruliferous and nonviruliferous aphids. In total, 1266 DEGs were identified at the level of transcription with 980 DEGs being upregulated and 286 downregulated in viruliferous aphids. At the protein level, among the 18 DEPs identified, the number of upregulated proteins in viruliferous aphids was twice that of the downregulated DEPs. Enrichment analysis indicated that these DEGs and DEPs were mainly involved in epidermal protein synthesis, phosphorylation, and various metabolic processes. Interestingly, the expressions of a number of cuticle proteins and tubulins were upregulated in viruliferous aphids. Taken together, our study revealed the complex regulatory network between BrYV and its vector M. persicae from the perspective of omics. These findings should be of great benefit to screening key factors involved in the process of virus circulation in aphids and provide new insights for BrYV prevention via vector control in the field.

A pilot study demonstrating a non‐invasive method for the measurement of protein turnover in skin disorders: application to psoriasis

BackgroundPrevious studies of epidermal kinetics in psoriasis have relied on invasive biopsy procedures or the use of radioactive labels. We previously developed a non-invasive method for measuring keratin synthesis in human skin using deuterated water labeling, serial collection of tape strips and measurement of deuterium enrichment in protein by mass spectrometry. This powerful method can be applied to measure other skin proteins and lipids collected by tape stripping. Here, for the first time, we apply this technique to investigate the epidermal kinetics of psoriasis, the first step in defining a kinetic profile for normal skin versus activated or quiescent psoriatic skin.MethodsPsoriatic subjects were given 2H2O orally as twice-daily doses for 16–38 days. Affected and unaffected skin was sampled by tape stripping and washing (modified Pachtman method). Proteins were isolated from the tape strips by a method that enriches for keratin. Turnover times were determined by gas chromatography/mass spectrometry. Kinetic data were compared to transepidermal water loss (TEWL).ResultsDeuterium-labeled protein from lesional psoriatic skin appeared at the skin surface within 3–8 days of label administration, whereas labeled protein from non-lesional skin requires 10–20 days to appear. Psoriatic skin had similar rate of growth despite varying anatomic location. Proteins recovered from tape strips were identified by nanoscale liquid chromatography/tandem mass spectrometry. Isolated peptides were >98% from keratin in uninvolved skin and >72% keratin in psoriatic skin. Revealing that one-quarter of all newly synthesized proteins in psoriatic skin are antimicrobial defense and other immune-related proteins. TEWL values were greater in lesional than non-lesional skin, suggesting barrier compromise in psoriatic skin despite increased clinical thickness.ConclusionsThis simple, elegant, and non-invasive method for measuring epidermal protein synthesis, which can also be adapted to measure epidermal lipids, provides a metric that may reveal new insights into the mechanisms and dynamic processes underlying psoriasis and may also provide an objective scale for determining response to therapeutic agents in pre-clinical and clinical trials. This opens a pathway to the non-invasive study of kinetics of protein formation in psoriasis or other skin diseases.

Measurement of protein metabolism in epidermis and dermis.

We found that, in the rabbit ear, the dermal protein contains 75.5% of cutaneous phenylalanine and 97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and 2.1% of proline are in the epidermal protein. This finding led us to develop two novel models that use phenylalanine and proline tracers and the rabbit ear to quantify protein kinetics in the epidermis and dermis. The four-pool model calculates the absolute rates of protein kinetics and amino acid transport, and the two-pool model calculates the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein synthesis was 93.4 +/- 37.6 mg x 100 g(-1) x h(-1), which was 10-fold greater than that of the dermal protein (9.3 +/- 5.8 mg x 100 g(-1) x h(-1)). These synthetic rates were in agreement with those measured simultaneously by the tracer incorporation method. Comparison of the four-pool and two-pool models indicated that intracellular cycling of amino acids accounted for 75 and 90% of protein kinetics in the dermis and epidermis, respectively. We conclude that, in the skin, efficient reutilization of amino acids from proteolysis for synthesis enables the maintenance of protein mass in the postabsorptive state.

Synthesis and distribution of cytokeratins in healthy and ulcerated bovine claw epidermis.

Keratinization of the epidermal cells of the bovine claw generates the horn that gives the tissue its mechanical strength. Disruption of keratinization is likely to have a detrimental effect on the strength and integrity of the horn, and could lead to solar lesions and lameness. As part of a wider investigation of the cell biological causes of lameness in dairy animals, we have compared keratin synthesis and distribution in healthy bovine claw tissue with those in hooves with solar ulcers. Protein synthesis was measured by [35S]-labelled amino acid incorporation in claw tissue explant cultures. [35S]-labelled protein synthesis was higher in tissue from diseased claws than in healthy claws, and highest at the ulcer site. The identity of proteins synthesised in vitro did not differ between healthy and diseased tissue. DNA synthesis indicative of cell proliferation was also elevated in diseased tissue. Immunoblotting after one- or two-dimensional electrophoresis showed cytokeratins (CK) 4, 5/6, 10 and 14 to be amongst those expressed in healthy claw tissue. The relative abundance of these keratins was not altered in healthy regions of ulcerated hooves, nor at the ulcer site, but CK16, not usually found in healthy tissue, was detected in the sole of diseased claws. CK5/6 and CK14 were shown by immunohistochemistry to be present in the basal epidermis of healthy tissue, whereas CK10 was found in supra-basal layers. In healthy tissue from ulcerated claws, this distribution was unaltered, but at the site of solar ulcers, CK5/6 and CK14 were each found in both basal and supra-basal epidermis. The study suggests that solar ulceration of the bovine claw is not associated with gross alteration in the keratin composition of the tissue, but causes abnormal distribution of cytokeratins, perhaps as a result of loss of positional cues from the basement membrane. Ulceration did, however, stimulate cell repair involving epidermal protein synthesis (including keratins), and keratinocyte proliferation.

Keratin: a journey of three decades.

During the past three decades, major progress has been made in our understanding of the processes which lead to the formation of a keratinized epidermis in normal and pathologic situations. Stimulated by clinical studies of exfoliative dermatitis and related diseases, a series of investigations have been performed which proved initially that the pathways and controls of epidermal protein synthesis were equivalent to protein synthetic pathways in all other tissues of the body. Keratin was identified as not only an insoluble protein which makes up the vast majority of the intracellular protein in the stratum corneum, but as a member of the intermediate filament family of cytoskeletal proteins. Of all such proteins, the keratins are most complex, occurring in two families separable on the basis of size, structure and isoelectric point. The keratin intermediate filaments are heteropolymers of two paired components, one from each family. The pairs of keratins which form the intermediate filaments in basal and differentiated layers of epidermis and other epithelia have been defined and antibodies to specific keratins are now being used for diagnostic purposes. Sophisticated biochemical, physicochemical, and molecular biologic studies have led to complete definitions of almost all the epithelial keratin molecules and to cloning of their genes. These genes are currently being used in analyses of control of keratin expression and definition of the specific abnormalities associated with "keratinopathies" including epidermolysis bullosa simplex and epidermolytic hyperkeratosis.

Effect of tumor promoting phorbol ester TPA on epidermal protein synthesis: Stimulation of an elongation factor 2 phosphatase activity by TPA in vivo

Topical application of the phorbol ester TPA to mouse skin causes an increase in the amount of elongation factor 2 (EF-2), a factor in eukaryotic protein synthesis, in the epidermal cytosol (2- to 3-fold) and particulate fraction (7-fold). Furthermore, as a consequence of this TPA treatment the activity of an epidermal EF-2 phosphatase is stimulated. The EF-2 phosphatase has an apparent molecular weight of around 38,000 daltons. The enzyme activity is induced as early as 45 minutes after TPA treatment and remains at the elevated level for more than 17 hours. Both of the TPA-induced effects result in an increase in unphosphorylated, i.e. active EF-2 and can be suppressed by cyclosporine A.

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either3H-leucine or35S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition. In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated35S-methionine. Thus they may be synthesized as proproteins. Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.

Hyperthermia and polyamine biosynthesis: Decreased ornithine decarboxylase induction in skin and kidney after heat shock

The effect of hyperthermia treatments on ornithine decarboxylase (ODC) induction in mouse tissue was determined both ` in ` vitro and ` in ` vivo . ` In ` vitro , the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to adult mouse skin pieces incubated at 37°C in serum-free MEM led to a dramatic increase in epidermal ODC activity 5 hours following treatment. In contrast, incubation temperatures of 40°C for the entire 5 hour incubation period rendered the skin pieces unresponsive to TPA for ODC induction. This inhibition of ODC induction was not the result of thermal skin kill, inactivation of TPA, or a general effect on epidermal protein synthesis. The inhibition of ODC induction could be reversed by switching the incubation temperature back to 37°C. ` In ` vivo , raising the core body temperature in male mice to 41°C for 1 hour resulted in a 78% decrease in kidney ODC activity. The kidney DNA synthesis and protein synthesis remained unaltered following the whole body hyperthermia treatments.

Epidermal protein synthesis in newborn rat skin.

Protein synthesis in newborn rat epidermis was assessed by in vivo labeling of epidermal proteins with radioactive glycine and the isolation of neutral buffer, sodium-dodecylsulfates (SDS) dithiothreitol soluble proteins, and 0.1 M NaOH soluble protein fractions of the epidermis. There was an increase in the labeling of proteins in the buffer and SDS fractions for 5 h after injection and then no further increase at 19 h after injection. In the 0.1 M NaOH soluble proteins and the final pellet after these extractions there was a progressive increase in glycine incorporation up to 19 h after injection. SDS gel electrophoresis of the labeled proteins showed early synthesis and continued synthesis of proteins of 56,000 and 65,000 daltons corresponding to the main fibrous proteins (alpha-keratins) of the epidermis. At 19 h a new protein band of 45,000 daltons first appeared and was associated with a decrease in the labeling of very high molecular weight (or highly aggregated) proteins. This later protein is presumptively identified as keratohyalin. Free amino acid pools were associated with both the neutral buffer and SDS extractable fractions.

Products of epidermal protein synthesis

The products of epidermal protein synthesis in tissue slice and cell-free systems have been characterized by chromatographic, electrophoretic, and immunologic methods. Radiolabeled proteins of heterogeneous size are found in the supernatant and particulate fractions from both systems. The major labeled proteins in the supernatant fraction have molecular weights of approximately 43 000 and 65 000. The natively-insoluble proteins of the particulate fraction were solubilized either with desoxycholate or alkaline urea. Subsequent analysis of theses fractions yielded a labeled, high molecular weight, urea-soluble protein whose electrophoretic mobility was slower than that of the heavy chain of myosin. The immunologic reaction between the radiolabeled fraction and antibody raised against filamentous protein suggests that synthesis of proteins identical to epidermal filamentous protein occurs in the in vitro systems.

Cell-Free Protein Synthesis in Mammalian Skin

Previous reports from our laboratory have described epidermal protein synthesis in an in vitro slice system (1, 2). This system has been used to localize the site of protein synthesis in epidermis (1) and to study the effects of several therapeutic agents upon epidermal macromolecular metabolism (2). Because of the limitations of studies with tissue slices, we have found it necessary to develop a cell-free protein synthesizing system from mammalian skin.

Studies of Epidermal Protein Metabolism: I. Incorporation of Amino Acids In Vivo

Previous studies of mammalian epidermal and hair proteins have either focused upon the physico-chemical (1) and morphological structure (2) of these unique entities or have been aimed at the characterization of the solubilized products of keratin breakdown following treatment with various protein denaturing agents (3). The processes of epidermal protein synthesis and keratin formation cannot be completely understood, however, until the biochemical methods now available for the study of protein synthesis (4) have been applied to the investigation of the epidermis. As a portion of a project aimed at reinvestigating epidermal protein metabolism, a study of the in vivo incorporation of radioac-tively labelled amino acids into epidermal proteins has been completed. The purpose of this report is to present this initial data.