New Functional Aspects of Cathepsin D and Cathepsin E

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.     

Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia

Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [³⁵S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A₁, a specific inhibitor of vacuolar-type H⁺-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.     

Emerging roles of cathepsin E in host defense mechanisms

Cathepsin E is an intracellular aspartic proteinase of the pepsin superfamily, which is predominantly expressed in certain cell types, including the immune system cells and rapidly regenerating gastric mucosal and epidermal keratinocytes. The intracellular localization of this protein varies with different cell types. The endosomal localization is primarily found in antigen-presenting cells and gastric cells. The membrane association is observed with certain cell types such as erythrocytes, osteoclasts, gastric parietal cells and renal proximal tubule cells. This enzyme is also found in the endoplasmic reticulum, Golgi complex and cytosolic compartments in various cell types. In addition to its intracellular localization, cathepsin E occurs in the culture medium of activated phagocytes and cancer cells as the catalytically active enzyme. Its strategic expression and localization thus suggests the association of this enzyme with specific biological functions of the individual cell types. Recent genetic and pharmacological studies have particularly suggested that cathepsin E plays an important role in host defense against cancer cells and invading microorganisms. This review focuses emerging roles of cathepsin E in immune system cells and skin keratinocytes, and in host defense against cancer cells. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.     

Increased Expression of Cathepsins E and D in Neurons of the Aged Rat Brain and Their Colocalization with Lipofuscin and Carboxy-Terminal Fragments of Alzheimer Amyloid Precursor Protein

Abstract: Age-related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Non-lysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age. Western blot analyses revealed that CE existed predominantly as the mature enzyme at 2 and 17 months of age, whereas it was present as not only the mature enzyme but also the proenzyme at 30 months of age. On the other hand, CD was mainly present in the mature form throughout development, although its level in these tissues was also significantly increased with aging. The CE-positive cortical and brainstem neurons of the aged rat corresponded well with cells emitting autofluorescence for lipopigments. By the double-staining technique, most of the CE-positive cortical and brainstem neurons of the aged rat were also positive for antibody to the carboxyl-terminal fragments of amyloid precursor protein (APP_634–695), intracellular accumulation of which is thought to be associated with age-related changes in the endosome/lysosome system. It is important that electron microscopy revealed that CE in brainstem neurons of the aged rat colocalized with CD in the lipofuscin-containing lysosomes. These results indicate that aging results in the increased expression and lysosomal localization of CE in cortical and brainstem neurons and changes in the endosomal/lysosomal proteolytic system, which may be related to lipofuscinogenesis and altered intracellular APP metabolism.     

Recombinant cathepsin E has no proteolytic activity at neutral pH

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.     

Gene expression profiling of mammary glands of cathepsin E-deficient mice compared with wild-type littermates

Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system and has been implicated in various physiological and pathological processes. Because of physiological substrates of cathepsin E have not yet been identified, however, the physiological significance of this protein still remains speculative. To better understand the physiological significance of cathepsin E in the mammary gland, we investigated the effect of the deficiency of this protein on the gene expression profile of the tissue. Here we used mammary glands derived from multiparous and non-pregnant 11-month-old syngenic wild-type (CatE(+/+)) and cathepsin E-deficient (CatE(-/-)) mice for extraction of total RNA from each tissue and subsequent mRNA amplification, DNA fragmentation, and hybridization with cDNA mixroarray chips. A total of 654 genes were identified as overexpressed (>2-fold) in CatE(-/-) mammary glands compared with CatE(+/+) counterparts. These included genes related to signal transduction, immune responses, growth factor activity, and milk proteins, which occupied a large portion of the gene fragments identified as overexpressed. In contrast, a total of 665 known genes were identified as underexpressed in the mammary gland of CatE(-/-) mice compared with CatE(+/+) counterparts. These included genes related to cytoskeleton, cell differentiation, cell cycle arrest and apoptosis, which occupied the majority of the gene fragments identified as underexpressed. The results thus suggest that cathepsin E in mammary glands plays a crucial role in the regulation of proteins involved in signaling, development, differentiation and proliferation in the mammary gland.     

Cathepsin K: A therapeutic target for bone diseases

Cathepsin K is a member of the papain family of cysteine proteases and is highly expressed in osteoclasts that mediate bone resorption. In this review, some of the known features of cathepsin K such as structure, function in bone resorption, gene regulation and its roles in physiological or pathophysiological processes are highlighted.     

Microglial Cathepsin B: An Immunological Examination of Cellular and Secreted Species

Abstract: The cysteine proteinase cathepsin B (CB) was isolated from immortalized murine BV-2 microglial cells and examined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to establish physicochemical properties of CB from what is generally considered the resident CNS macrophage. Microglial proteases have been implicated in several pathological processes occurring in the CNS, including neurodegeneration. Murine microglial CB was observed to consist of two major single-chain species of 32 and 34 kDa, with pls of 5.5-5.2 and 5.1-4.5, respectively. In addition, a minor 24-kDa CB species was also observed in some microglial preparations. The major CB isozymes in microglia differed from those observed in murine liver and brain, which consisted of both single- and double-chain CB variants of 31 and 24–25 kDa/5 kDa, respectively, with pl values of 5.5-4.5. A microglial pro-CB of 37 kDa was also isolated, which could be processed to the 34-kDa single-chain CB species. Cystatin was observed to prevent pro-CB processing, whereas E-64 and leupeptin were only partially inhibitory. The 37-kDa pro-CB species was observed to undergo processing into the 34-kDa CB species when incubated at pH 5.5 but remained stable with respect to molecular mass when incubated at pH 7.0. In contrast, the 34-kDa single-chain CB species was observed to autodegrade when incubated at pH 7.0, whereas incubation at pH 5.5 did not affect the integrity of the species as monitored by immunoblotting. Both pro-CB and 32-kDa single-chain CB species were observed extracellularly following lipopolysaccharide activation of BV-2 microglial cells.     

DNA vaccines: a mini review

DNA vaccines are a major breakthrough in the field of vaccination with several advantages over traditional vaccines. Unlike traditional vaccines, DNA vaccines stimulate both arms of the immune system offering long lasting immunity. DNA vaccines not only have the potential to fight against infectious diseases such as influenza and hepatitis but they can also be used to prevent autoimmune diseases such as multiple sclerosis. In general, this article is intended as a mini-review to discuss DNA vaccination, as well as patents on different types of DNA vaccines.     

Selective detection of Cathepsin E proteolytic activity

Background

Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide–substrate that is only sensitive to Cath E but not Cath D.

Methods

Substrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH₂, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence.

Results

Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (K_cat/K_M) of Cath E for substrate e was 16.7 μM⁻¹ S⁻¹. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B.

Conclusions

This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement.

General significance

This study forms the foundation of Cath E specific inhibitor development in further studies.     

组织蛋白酶B的新型天然抑制剂

组织蛋白酶B是木瓜蛋白酶类半胱氨酸蛋白酶家族的重要成员,它与人类多种疾病相关,尤其是在恶性肿瘤的侵袭转移过程中扮演了重要角色.通过随机筛选,发现了五个对组织蛋白酶B具有较好抑制活性的天然化合物prodelphinidin B-23'-O-gallate(1),prodelphinidin B-2(2),ImJcyarddin B-2(3),puexin A(4)和(-)epigallocatechin-3-O-gallate(5),其IC50值分别为0.58,0.44,0.76,2.07和0.96umol/L.这五个抑制剂为黄烷醇类化合物,均为组织蛋白酶B的新型天然抑制剂.     

Cathepsin E (EC 3.4.23.34)--a review

Cathepsin E belongs to the third class of enzymes - hydrolases, a subclass of peptide bond hydrolases and a sub-subclass of endopeptidases with aspartic catalytic sites. Cathepsin E is an endopeptidase with substrate specificity similar to that of cathepsin D. In a human organism, cathepsin E occurs in: erythrocytes, thymus, dendritic cells, epithelial M cells, microglia cells, Langerhans cells, lymphocytes, epithelium of gastrointestinal tract, urinary bladder, lungs, osteoclasts, spleen and lymphatic nodes. In human cells, loci of the gene of pre-procathepsin E are located on chromosome 1 in the region 1231-32. The catalytic site of cathepsin E is two residues of aspartic acid - Asp96 and Asn281, occurring in amino acid triads with sequences DTG96-98 and DTG281-283. To date, no particular role of cathepsin E in the metabolism of proteins in normal tissues has been found. However, it is known that there are many documented pathological conditions in which overexpression of cathepsin E occurs.     

动物组织蛋白酶L=like基因家族的进化分析

目的：组织蛋白酶L-like家族是在溶酶体中发现的一类非常重要的半胱氨酸组织蛋白酶。其主要功能为催化各种蛋白质的水解,并通过水解蛋白质参与到许多的生理调节过程当中。根据序列比对分析和传统的功能分类,在动物中,组织蛋白酶L．1ike家族成员包括组织蛋白酶L、V、S、K、H和F。但是这些家族成员之间的进化关系仍然没有详细研究分析清楚。本课题主要研究组织蛋白酶L-like家族成员之间的进化关系。方法：本研究通过搜集整理22个物种的177条组织蛋白酶L-1ike家族蛋白的序列,并构建系统发育进化树来分析组织蛋白酶L-like家族各成员之间的进化关系。结果：序列数据结果显示,串联重复在组织蛋白酶L-1ike家族的进化过程中发生。斑马鱼的组织蛋白酶L,爪蟾的组织蛋白酶S和K,大鼠和小鼠的组织蛋白酶L都发生过明显的串联重复事件。进化树结果显示了组织蛋白酶H、S和K、L和V之间的进化关系,组织蛋白酶S和K在脊椎动物出现的进化过程中,从组织蛋白酶L中分化出来,与他们在脊椎动物体内的特异性功能,以及脊椎动物在进化过程中分化产生的特异性功能相对应。结论：在物种进化的过程中,组织蛋白酶L-1ike家族成员F、H、S和K、L和V按时间顺序分化,这表明组织蛋白酶L-1ike基因家族结构和功能的分化与新的物种和新的功能出现密切相关。     

Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.     

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.     

Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases)

Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.