Epidermal differentiation: the role of proteases and their inhibitors

Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent findings on the role of cystatin M/E and legumain as a functional dyad in skin and hair follicle cornification, a paradigm example of the regulatory functions exerted by epidermal proteases, will be discussed.     

On the role of proteases, their inhibitors and the extracellular matrix in promoting neurite outgrowth

Using a novel method, a monoclonal antibody was produced which can directly block the activity of an extracellular matrix-associated neurite outgrowth promoting complex (Matthew and Patterson, 1983). Presumably binding at or near the active site, this antibody recognizes a determinant consisting of heparan sulfate and a larger molecule which is likely to be laminin (Matthew et al., in preparation). The antibody has been further used to localize this determinant in adult tissues in vivo. Extracellular binding is seen at sites known to promote axon regeneration in the peripheral nervous system and is not seen in the central nervous system (Matthew et al., in preparation). In investigating how neurons may modify their environment as they grow processes, we have recently found that sensory and sympathetic neurons spontaneously release a collagenase and a plasminogen activator from their distal processes and/or growth cones (Pittman, 1985). A 43 kD irreversible inhibitor of the plasminogen activator is secreted by cardiac myocytes and is found on the surfaces of cultured neurons (Pittman, 1984). This inhibitor is also released by nonneuronal cell cultures from peripheral, but not central, nerves (Pittman, unpublished). Of interest in relation to the proteoglycan neurite outgrowth promoting complex is the finding that the 43 kD inhibitor preparation binds heparin tightly and can displace laminin from its heparin binding site (Patterson and Pittman, unpublished). Thus it is possible that the protease/inhibitor system could affect outgrowth via interaction with the neurite outgrowth promoting complex in the extracellular matrix.     

Structural studies of cysteine proteases and their inhibitors.

Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.     

Activities and cellular localization of yeast proteases and their inhibitors

We have found that proteases A, B and C of $\text{Saccharomyces cerevisiae}$ are localized in the vacuole. The corresponding inhibitors of these three proteases are present in the extravacuolar cytosol. The compartmentation of the yeast proteases suggests that their function is primarily the intravacuolar digestion of proteins. Yearst cells were grown under various conlitions, and it was found that culture conditions which either induce biochemical differentiation of cells or which do not allow growth result in the enhancement of proteolytic activities.     

The ovarian cycle and control of ovulation

The ovarian cycle of primates is a sequence of events reflecting follicular growth and development, the ovulation of a mature oocyte and the formation of a functional corpus luteum. A typical cycle generally consists of three phases: (1) the follicular or proliferative phase, (2) ovulation and (3) the luteal or secretory phase. Within this general pattern exists considerable species variation in terms of cycle length, timing of ovulation, presence or absence of menstruation and influence of season.
Details of the basic physiological mechanisms controlling cyclic ovarian function in primates are known for only a few species. Concentrating on information derived from studies in women and in rhesus and marmoset monkeys, this paper examines some of the hormonal mechanisms underlying the primate ovarian cycle with particular reference to the factors controlling preovulatory follicular development during the follicular phase.     

The novel inhibitors of serine proteases

Thirty optically active nonprotein α-amino acids and peptides based thereon have been screened for their ability to interact with bovine trypsin and proteinase K from Tritirachium album Limber, which belong to the group of serine proteases. Both structure-based drug design approach and determination of enzyme activity have been used to identify low molecular weight inhibitors of trypsin and proteinase K. Compounds have been selected that according to the docking analysis were able to interact with trypsin and proteinase K. Following the docking analysis measurement of enzymes activity (2R,3S)-β-hydroxyleucine and (2S,3R)-β-hydroxyleucine inhibited both enzymes activity, whereas (S)-α-methyl-β-phenylalanine, (R)-α-methyl-β-phenylalanine, (S)-allylglycine, (R)-allylglycine, (S)-α-allylalanine, (R)-α-allylalanine and allo-O-ethylthreonine inhibited only proteinase K; and N-formyl-(S)-methionyl-(2S,3R)-hydroxyleucine, N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine, N-formyl-(S)-methionyl-(S)-allylglycine and N-formyl-(S)-methionyl-(R)-allylglycine inhibited trypsin. It has been shown that inhibition of trypsin by (2R,3S)-β-hydroxyleucine and N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine is of a competitive mode.     

The involvement of proteases and protease inhibitors in neovascularization

Bovine capillary endothelial cells have been found to respond to several stimuli by producing increased amounts of plasminogen activator and latent collagenase. These stimulators include the tumor promoter tetradecanoyl phorbol-13-acetate as well as crude preparations from a human hepatoma, bovine retinae, and mouse adipocytes, all of which are known to contain angiogenic factors. Endothelial cells and skin fibroblasts do not respond to these stimuli in the same way, indicating a specificity of the response. In addition, inhibitors of plasmin and vertebrate collagenase have been isolated from cartilage, a tissue resistant to neovascularization. We have proposed that these specific protease inhibitors confer on cartilage its antiangiogenic properties.     

Exocellular proteases of Malbranchea gypsea and their role in keratin deterioration

Malbranchea gypsea IMI 338168 isolated from the soils of Keoladeo National Park, Bharatpur was studied for its ability to produce exocellular proteases on glucose – gelatin medium at pH 7; 28°C. The fungus was observed to be a potent producer of such enzymes. Protease production was optimal at 15 days of incubation. Asparagine was repressive to protease expression. No relationship existed between the amount of enzyme production and increase in biomass. Exogenous sugars suppressed enzyme production in descending order as follows: glucose > mannose > maltose > arabinose > fructose. The enzymes expressed showed the ability to degrade three keratinous substrate tested. Buffalo skin was the most actively degraded substrate when exogenous glucose was present, and was also the most resistant to degradation in the absence of glucose. The rate of keratin deterioration was independent of enzyme activity. Production of protease enzymes especially keratinases is ecologically important in a place like a National Park because such enzymes degrade keratinous detritus derived from mammals and birds. Accumulation of such materials can be a cause of pollution and can provide a breeding spot for various types of pathogens. This revised version was published online in June 2006 with corrections to the Cover Date.     

Caspase-like proteases and their role in programmed cell death in plants

The investigations performed over recent few years have proved the existence of caspase-like proteases in plants. Three groups of caspase-like proteases: metacaspases, legumain family proteases (VPEs) and saspases have been identified and characterized in plants so far. A considerable amount of evidence supports the role of these enzymes in programmed cell death (PCD) occurring during plant development, their organ senescence as well as hypersensitive response (HR) after pathogen attack. Current knowledge of these enzyme molecular and biochemical structures is summarized in the paper. The homology of caspase-like proteases to animal caspases has been also indicated. Some future perspectives of research concerning the signal pathway during PCD, the regulation of activity and mode of action of these proteases are presented in the article.     

Defense against own arms: staphylococcal cysteine proteases and their inhibitors

Staphylococcus aureus is a human pathogen causing a wide range of diseases. Most staphylococcal infections, unlike those caused by other bacteria are not toxigenic and very little is known about their pathogenesis. It has been proposed that a core of secreted proteins common to many infectious strains is responsible for colonization and infection. Among those proteins several proteases are present and over the years many different functions in the infection process have been attributed to them. However, little direct, in vivo data has been presented. Two cysteine proteases, staphopain A (ScpA) and staphopain B (SspB) are important members of this group of enzymes. Recently, two cysteine protease inhibitors, staphostatin A and staphostatin B (ScpB and SspC, respectively) were described in S. aureus shedding new light on the complexity of the processes involving the two proteases. The scope of this review is to summarize current knowledge on the network of staphylococcal cysteine proteases and their inhibitors in view of their possible role as virulence factors.     

Lysosomal cysteine proteases: structural features and their role in apoptosis

Among the variety of proteolytic enzymes enormous progress has been seen recently in our understanding of lysosomal cysteine proteases, also known as cysteine cathepsins. These enzymes play a crucial role in diverse biological processes in physiological and pathological states, including genetic diseases. In the present review, their properties and structural features that are important to an understanding of their biological function are presented. Special emphasis is given to the newly discovered role of lysosomal cathepsins in apoptotic pathways.     

Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration

Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein‐labeled peptide chloromethyl ketone that specifically binds to trypsin/thrombin‐like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin‐like activity was localized, using the fluorescein‐conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin‐like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin‐like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin‐like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin‐like activity by N‐tosyl‐l ‐phenylalanine chloromethyl ketone and chymostatin as well as trypsin‐like activity by N‐tosyl‐l ‐lysine chloromethyl ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.     

The role of plasminogen activator in ovulation

The role of plasminogen activator in ovulation was investigated using the inhibitor, trans-aminomethylcyclohexane carboxylic acid (t-AMCHA). In the regular cycle rat, the plasminogen activator activity of the follicles increased from the diestrus to the estrus phase. In the latter phase, a proteolytic enzyme which was not inhibited by t-AMCHA appeared. After ovulation, the plasminogen activator activity decreased. When ovulation was induced in immature rats by pregnant mare serum gonadotrophin and human chorionic gonadotrophin, remarkable fibrinolytic activity appeared in the ovaries immediately before ovulation. When t-AMCHA was given in the ovulation-induced rats, the fibrinolytic activity of the ovaries was suppressed, the number of ovulated ova decreased and the timing of ovulation was delayed. When t-AMCHA solution was given to rats in the proestrus phase, ovulation was almost completely suppressed, but aprotinin solution exerted no effect on ovulation. These results suggest that plasminogen activator is a key enzyme in ovulation, and that the chain reaction from plasminogen activator to proteolytic enzyme (including collagenase) is of greater importance than that of plasminogen activator to plasmin.     

血纤维蛋白溶酶原激活因子及其抑制因子在神经系统的作用

血纤维蛋白溶酶原激活因子和血纤维蛋白溶酶原激活因子的抑制因子与神经肌肉系统的生长发育有十分密切关系。ＰＡ对神经细胞的增殖、迁移、神经的变性与再生和对肌肉卫星细胞的融全以及对神经肌肉间突触的成和可塑性都有明显的作用。     

Reversible detection of proteases and their inhibitors by a pulsed chronopotentiometric polyion-sensitive electrode

Polymer membrane electrodes operated by pulsed chronopotentiometry have recently been introduced to replace traditional ion-selective electrodes for a number of applications. While ion-selective electrodes for the polycation protamine have been reported, for instance, a pulsed chronopotentiometric readout mode (called here pulstrode) provides improved stability and reproducibility while exhibiting sufficient selectivity for the direct detection of protamine in undiluted whole blood samples. Here, such protamine-sensitive pulstrodes are applied for the real-time detection of the activity of the protease trypsin and its soybean inhibitor. This is possible because small fragments produced by the trypsin digestion are not detectable by the protamine-sensing membrane. The real-time response to the proteolytic reaction is shown to exhibit good reproducibility and reversibility, and the initial reaction rate is dependent on the concentration of the protease and its inhibitor.