半胱氨酸蛋白酶拟肽抑制剂设计新进展

半胱氨酸蛋白酶包括多种酶,这些酶在广泛的生命过程中发挥作用。人类正常的半胱氨酸蛋白酶表达失调,寄生虫、病毒的半胱氨酸蛋白酶表达与多种病理情况相关。对于这类疾病,抑制半胱氨酸蛋白酶是一个可行的药物治疗策略。当前这类药物设计的目标是3种结构不同的半胱氨酸蛋白酶,即木瓜蛋白酶家族、半胱氨酸-天冬氨基特异性蛋白酶家族(caspases)和小核糖核酸病毒科半胱氨酸蛋白酶抑制剂家族。本文综述了近年来有关半胱氨酸蛋白酶抑制剂的设计思路。     

溶酶体组织蛋白酶B、L和S在糖尿病肾病发生发展中的作用

溶酶体组织蛋白酶(cathepsins, Cats)是一类存在于细胞内和细胞外的具有多种生物活性的蛋白酶,是哺乳动物体内蛋白水解的主要参与者。除了能影响细胞外基质稳态、自噬、细胞凋亡过程外,还对肾小球通透性、内皮功能和炎症具有调节作用。多种Cats表达活性的失调参与急慢性肾脏疾病的发生发展。其中溶酶体组织蛋白酶B(cathepsins B, CatB)、溶酶体组织蛋白酶L(cathepsins L, CatL)及溶酶体组织蛋白酶S(cathepsinsS, CatS)作为糖尿病肾病(diabetic nephropathy, DN)病理生理中的关键参与者受到了广泛的关注。目前,越来越多的证据表明CatB、L和S可能是诊断和治疗DN的新靶点。本文主要对CatB、L及S在DN的作用及相关机制进展进行综述。     

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.     

活素与肿瘤的关系

活素(livin)是近年发现的IAP家族的新成员之一,存在于胎儿及成人的多种组织中。活素在很多肿瘤组织中高表达,其抗细胞凋亡机制与胱天蛋白酶(caspases)蛋白家族和线粒体内Smac相关。由于在多数人类常见的肿瘤组织都有活素的高表达,故可以作为一种新的标志物应用于肿瘤的临床诊断与治疗。     

马拉色菌致病相关因子的研究进展

马拉色菌是人类和温血动物皮肤的常驻菌,亦是一种条件致病性真菌,它可以引起花斑癣、马拉色菌毛囊炎、脂溢性皮炎等多种疾病。脂酶、蛋白酶、磷脂酶和脂氧合酶等为马拉色菌主要侵袭性酶。马拉色菌与角质形成细胞共培养可引起角质形成细胞多种形态学改变、细胞因子含量变化和细胞凋亡。     

基质金属蛋白酶及其组织抑制剂研究进展

基质金属蛋白酶家族是细胞外基质降解过程中的重要酶类,组织金属蛋白酶抑制剂是基质金属蛋白酶的天然抑制物。研究证实,细胞外基质中基质金属蛋白酶及其组织抑制剂的失衡与多种病理机制有关,尤其与肿瘤的侵袭和转移密切相关。本就基质金属蛋白酶及其组织抑制剂的性质、结构以及功能进行了综述。     

一类新型的抗病毒药物:病毒进入抑制剂

艾滋病是严重威胁人类健康的病毒性传染病.目前临床上抗人类免疫缺陷病毒(HIV)感染的药物主要是针对反转录酶和蛋白酶.反转录酶抑制剂和蛋白酶抑制剂的联合使用能显著降低HIV感染者的发病率和病死率,然而不良反应较大,价格昂贵,而且耐药的问题日益突出.近来一类新型抗HIV药物相继问世,其中T-20已经被美国食品药品管理局(FDA)批准正式在临床使用,此外几十种同类药物已经进入临床试验.     

白念珠菌致病相关的水解酶

白念珠菌是一种重要的人类致病性真菌,其致病机制与多种因素有关.水解酶是白念珠菌最重要的毒力因子之一,在其入侵宿主过程中起关键作用.白念珠菌水解酶包括分泌型天冬氨酸蛋白酶、磷脂酶和脂肪酶,介导白念珠菌的表型转换、对宿主组织的黏附及对宿主免疫系统的干预,使其能够入侵宿主组织和逃避宿主的免疫防御机制.该文我们综述了白念珠菌水解酶的生物学属性和致病机制的研究进展.     

家蚕组织蛋白酶基因家族的鉴定及表达特征分析

家蚕是鳞翅目完全变态昆虫,在其变态过程中伴随着巨大的形态变化,包括旧组织的解离和新组织的形成,在这过程中有多种组织蛋白酶参与。组织蛋白酶是一类细胞内蛋白酶,广泛存在于各个物种中,包括组织蛋白酶B、H、L等几个亚家族。对家蚕组织蛋白酶的研究将有利于阐明家蚕变态发育的详细过程。通过对家蚕基因组数据库进行筛选,共在家蚕中鉴定到13种组织蛋白酶,并对这13种组织蛋白酶的基本信息和表达模式进行了分析。另外,利用家蚕基因芯片数据和荧光定量PCR分析,鉴定编号为BGIBMGA004622的基因为卵巢特异表达的组织蛋白酶L亚家族基因。该基因全长1 209 bp,编码402个氨基酸。经过序列分析,该酶与其他物种的组织蛋白酶L具有较高的同源性,其活性位点高度保守,且与鳞翅目的组织蛋白酶L在进化上聚为一支。同时,对该基因进行克隆并原核表达,结果显示重组蛋白以包涵体的形式表达。定量PCR结果显示,该酶在蛹发育初期表达量逐渐升高,至蛹3 d达到最高值,推测其可能参与卵巢与卵母细胞的发育过程。     

CD147的研究进展

CD147分子是一种广泛表达于人体多种组织的跨膜糖蛋白,属于免疫球蛋白超家族。CD147在多种肿瘤细胞和组织中高表达,通过诱导基质金属蛋白酶(MMP)的分泌促进了肿瘤的浸润、转移。同时,CD147与炎症反应如类风湿性关节炎、动脉粥样硬化,以及细胞、组织的分化和发育等密切相关。简要综述了CD147参与的多种生理、病理过程。     

Cysteine cathepsins in human cancer

Proteases play causal roles in the malignant progression of human tumors. This review centers on the roles in this process of cysteine cathepsins, i.e., peptidases belonging to the papain family (C1) of the CA clan of cysteine proteases. Cysteine cathepsins, most likely along with matrix metalloproteases (MMPs) and serine proteases, degrade the extracellular matrix, thereby facilitating growth and invasion into surrounding tissue and vasculature. Studies on tumor tissues and cell lines have shown changes in expression, activity and distribution of cysteine cathepsins in numerous human cancers. Molecular, immunologic and pharmacological strategies to modulate expression and activity of cysteine cathepsins have provided evidence for a causal role for these enzymes in tumor progression and invasion. Clinically, the levels, activities and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. Understanding the roles that cysteine proteases play in cancer could lead to the development of more efficacious therapies.     

Comprehensive search for cysteine cathepsins in the human genome

Our study was aimed at examinating whether or not the human genome encodes for previously unreported cysteine cathepsins. To this end, we used analyses of the genome sequence and mRNA expression levels. The program TBLASTN was employed to scan the draft sequence of the human genome for the 11 known cysteine cathepsins. The cathepsin-like segments in the genome were inspected, filtered, and annotated. In addition to the known cysteine cathepsins, the scan identified three pseudogenes, closely related to cathepsin L, on chromosome 10, as well as two remote homologs, tubulointerstitial protein antigen and tubulointerstitial protein antigen-related protein. No new members of the family were identified. mRNA expression profiles for 10 known human cysteine cathepsins showed varying expression levels in 46 different human tissues and cell lines. No expression of any of the three cathepsin L-like pseudogenes was found. Based on these results, it is likely that to date all human cysteine cathepsins are known.     

Human cathepsins F and W: A new subgroup of cathepsins.

Human cathepsin F is a recently described papain-like cysteine protease of unknown function. To investigate the evolutionary relatedness to other human cathepsins, we determined the genomic organization and the chromosomal localization of cathepsin F and isolated its putative promoter region. The gene of human cathepsin F (CTSF) is composed of twelve exons and eleven introns and was found to be similar to that of cathepsin W but different from the cathepsins K, S, L, O, B, and C. The splice sites of nine out of the eleven introns were identical to those determined in the cathepsin W gene (CTSW), whereas introns one and ten were unique for CTSF. The 4. 7 kb gene was mapped to the long arm of chromosome 11 at position q13.1-3, a locus shared with CTSW. Phylogenetic analysis of human cathepsin protein sequences demonstrated that (i) cathepsins F and W are evolutionarily separated from other human cathepsins, and (ii) cysteine proteases closely related to human cathepsin W and F are also expressed in parasites and mammals. Based on these phylogenetic findings, on the presence of a particular protein motif ("ERFNAQ") in the propeptides of cathepsins F and W as well as the genomic organization and chromosomal localization of their genes, we concluded that F and W form a novel subgroup of cathepsin proteases. We suggest the naming "cathepsin F-like" proteases distinct from the previously described cathepsins "L- and B-like" subgroups.     

Multiple Roles for Cysteine Cathepsins in Cancer

Cysteine cathepsins are a family of proteases that have recently emerged as important players in cancer, and have variously been reported to be involved in apoptosis, angiogenesis, cell proliferation, and invasion. In normal cells, cysteine cathepsins are typically localized in lysosomes and other intracellular compartments, and are involved in protein degradation and processing. However, in certain tumors, cathepsins are translocated from their intracellular compartments to the cell surface, and can even be secreted. In addition, the expression and activity levels of some cysteine cathepsins are upregulated in human and mouse cancers. Understanding which cathepsins are critically involved, what their substrates are, and how they may be mediating these complex roles in cancer are important questions to address. We highlight recent results that begin to answer some of these questions, illustrating in particular the lessons from studying a mouse model of multistage carcinogenesis, which suggests distinctive roles for individual cysteine cathepsins in tumor progression.     

Tumor cell- and microenvironment-specific roles of cysteine cathepsins in mouse models of human cancers

The human genome encodes for 11 papain-like endolysosomal cysteine peptidases, collectively known as the cysteine cathepsins. Based on their biochemical properties and with the help of experiments in cell culture, the cysteine cathepsins have acquired a reputation as promotors of progression and metastasis of various cancer entities. However, tumors are known to be complex tissues in which non-cancerous cells are also critical for tumorigenesis. Here we discuss the results of the intense investigation of cathepsins in mouse models of human cancers. We focus on models in immunocompetent mice, because only such models allow for analysis of cathepsins in a fully functional tumor microenvironment. An important outcome of those studies was the identification of cancer-promoting cathepsins in tumor-associated macrophages. Another interesting outcome of these animal studies was the identification of a homeostatic tumor-suppressive role for cathepsin L in skin and intestinal cancers. Taken together, these in vivo findings provide a basis for the use of cysteine cathepsins as therapeutic targets, prodrug activators, or as proteases for imaging tumors.     

Evolution of placentally expressed cathepsins

Species and strain variants of a family of placentally expressed cathepsins (PECs) were cloned and sequenced in order to identify evolutionary conserved structural characteristics of this large family of cysteine proteases. Cathepsins M, P, Q, and R, are conserved in mice and rats but homologs of these genes are not found in human or rabbit placenta, showing that this family of proteases are probably restricted to rodents. Species-specific gene duplications have given rise to variants of cathepsin M in mice, and cathepsin Q in rats. Although the PECs have diverged at a greater rate than the other lysosomal cathepsins, residues around the specificity sub-sites of the individual enzymes are conserved. Strain-specific polymorphisms show that the evolutionary rate of divergence of cathepsins M and 3, the most recently duplicated pair of mouse genes, is even higher than the other PECs. In human placenta, critical functions of the PECs are probably performed by broader specificity proteases such as cathepsins B and L.     

Human cysteine cathepsins are not reliable markers of infection by Pseudomonas aeruginosa in cystic fibrosis

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps-) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (～2-fold) in the Ps+ and Ps- groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps- samples, despite the possible release of the ～31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection.     

Mouse cathepsin M, a placenta-specific lysosomal cysteine protease related to cathepsins L and P

The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species.     

Cysteine cathepsins B,X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.     

Cysteine cathepsin non-inhibitory binding partners: modulating intracellular trafficking and function

Cysteine cathepsins play a fundamental role in tumor growth, invasion and migration, angiogenesis, and the metastatic cascade. Evidence of their overexpression in a wide array of human tumors has been well documented. Cysteine cathepsins seem to have a characteristic location-function relationship that leads to non-traditional roles such as those in development and pathology. For example, during tumor development, some cysteine cathepsins are found not just within lysosomes, but are also redistributed into presumptive exocytic vesicles at the cell periphery, resulting in their secretion. This altered localization contributes to non-lysosomal functions that have been linked to malignant progression. Mechanisms for altered localization are not well understood, but do include the interaction of cysteine cathepsins with binding partners that modulate intracellular trafficking and association with specific regions on the cell surface.