Transthyretin and cystatin C are catabolized in proximal tubular epithelial cells and the proteins are not useful as markers for renal cell carcinomas

Ten human kidney specimens and thirty-two renal cell carcinomas were investigated for the presence of transthyretin mRNA and cystatin C mRNA using Northern blot analysis. Five of ten kidney specimens and 15 of 32 renal carcinomas were also immunohistochemically investigated for the presence of the corresponding proteins. Transthyretin mRNA could not be detected in any of the normal or neoplastic tissue preparations, whereas low amounts of cystatin C mRNA were found in nine of ten normal kidneys and in 24 of 32 renal cell carcinomas. Immunoreactive transthyretin and cystatin C were present in proximal tubular epithelial cells of all kidney specimens, whereas neither of the proteins was detected in the tumour cells of the renal carcinomas. Immunoreactive cystatin C was, however, demonstrated in scattered monocyte/macrophage-like cells. We conclude that the presence of immunoreactive transthyretin and cystatin C in proximal tubular cells of the kidney is most likely due to reabsorption of the proteins from the primary urine. The small amounts of cystatin C mRNA in some of the normal and neoplastic renal preparations are probably due to cystatin C synthesis in macrophages. Transthyretin has been recommended as an immunohistochemical marker for renal cell carcinomas. Our results, however, clearly indicate that neither transthyretin nor cystatin C constitutes a useful marker for such neoplasms.     

Structural and Binding Studies of SAP-1 Protein With Heparin

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (K_D = 158 nm ). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity.     

Cysteine cathepsins: regulators of antitumour immune response

Cysteine cathepsins are lysosomal cysteine proteases that are involved in a number of important biological processes, including intracellular protein turnover, propeptide and hormone processing, apoptosis, bone remodelling and reproduction. In cancer, the cathepsins have been linked to extracellular matrix remodelling and to the promotion of tumour cell motility, invasion, angiogenesis and metastasis, resulting in poor outcome of cancer patients; however, cysteine cathepsins are also involved at different levels of the innate and adaptive immune responses. Their best known role in this aspect is their contribution to major histocompatibility complex class II antigen presentation, the processing of progranzymes into proteolytically active forms, cytotoxic lymphocyte self-protection, cytokine and growth factor degradation and, finally, the induction of cytokine expression and modulation of integrin function. This review is focused on the role of cysteine cathepsins in the antitumour immune response and the evaluation of their pro- and anticancer behaviours during the regulation of these processes.     

Size and morphology of toxic oligomers of amyloidogenic proteins: a case study of human stefin B

Amyloid-induced toxicity is a well-known phenomenon but the molecular background remains unclear. One hypothesis relates toxicity to amyloid–membrane interactions, predicting that amyloid oligomers make pores into membranes. Therefore, the toxicity and membrane interaction of prefibrillar aggregates and individual oligomers of a non-pathological yet highly amyloidogenic protein human stefin B (cystatin B) was examined. By monitoring caspase-3 activity and by testing cell viability, we showed that the lag phase aggregates obtained at pH 5 and 3 were toxic to neuroblastoma cells. Of equal toxicity were the higher-order oligomers prepared at pH 7 by freeze–thaw cycles. The higher-order oligomers eluted on size-exclusion chromatography (SEC) as a broad peak comprising hexamers, octamers, 12- and 16-mers, well separated from monomers, dimers and tetramers. Only oligomers higher than the tetramers (Rh >3.5 nm) proved toxic, in contrast to dimers and tetramers. In accordance with data from SEC, dynamic light scattering and atomic force microscopy data indicate that the toxic oligomers have diameters larger than 4 nm. Critical pressure measurements showed that the toxic higher-order oligomers inserted more effectively into model lipid monolayers than dimers and tetramers. They also bound, similarly to prefibrillar aggregates, to the plasma membrane and became internalized. Taken together, our results confirm the importance of membrane interaction and perforation in the phenomenon of cytotoxicity.     

The possible place of cathepsins and cystatins in the puzzle of Alzheimer disease

Lysosomal proteinases (cathepsins) and their endogenous inhibitors (cystatins) have been found to be closely associated with senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles in Alzheimer disease (AD). Further, profound changes in the lysosomal system seem to be an early event in “at-risk” neurons of AD brains. There is an ongoing controversy as to whether lysosome-associated proteolytic mechanisms are causally related to the development and/or further progression of the disease. The present article deals with some arguments “pro” and “contra” an involvement of the endosomal/lysosomal pathway in amyloidogenesis as a cardinal process in AD. Other putative targets of acidic proteinases and their natural inhibitors in the pathogenesis of AD (such as formation of neurofibrillary tangles and regulation of apolipoprotein E) are also discussed.     

Cystatin D, a natural salivary cysteine protease inhibitor, inhibits coronavirus replication at its physiologic concentration

This study was conducted to examine the effect of cystatin D, a newly discovered salivary cysteine protease inhibitor, on human coronavirus replication. When MRC-5, human diploid lung cells, were incubated with dilutions of recombinant human cystatin D from 0.65–10 μM for 1 h prior to, during and after infection with coronavirus OC43 and 229e strains, a decrease in virus yield was observed resulting in an IC₅₀ of 0.8 μM for both virus strains. This dose is within the normal concentration range of cystatin D, 0.12–1.9 μM found in saliva. When a single dose, 2.5 μM, was applied, cystatin inhibition of release of virus progeny was not overcome until three days post infection whereas inhibition by leupeptin, a serine and cysteine protease inhibitor, was completely abrogated by two days. When cellular toxicity was measured by ³H-thymidine uptake, cystatin D did not markedly affect cell proliferation below a 10 μM dose. The results demonstrate that cystatin D is a potent inhibitor of coronavirus replication.     

Cysteine protease inhibitory activity and levels of salivary cystatins in whole saliva of periodontally diseased patients

The 3 human salivary cystatins S, SA and SN are multifunctional proteins that possess a cysteine protease inhibitory property, but their ability to act as such is very different (SN > SA > S). One form, S, also appears to possess antibacterial properties towards the bacterium Porphyromonas gingivalis, often associated with periodontal diseases. In this study we measured the total cystatin inhibitory activity and the levels of each salivary cystatin in the whole saliva of 8 periodontally diseased patients and 2 groups of control subjects (n = 6 and n = 10). The total cystatin inhibitory activity and the total salivary cystatin concentration in the periodontally diseased patients were found to be lower than the controls (p < or = 0.005). The concentration of S was depleted to levels that would not allow it to be an effective antibacterial agent, and the concentration of SA, although depleted in some cases, was still present at sufficient levels to allow it to act as an effective physiological inhibitor of cathepsin L. The concentration of cystatin SN was also depleted in the periodontally diseased patients, but was still present in sufficient quantities to act as an effective physiological cysteine protease inhibitor of cathepsins H and L. In comparison, the concentration of all 3 salivary cystatins in the control subjects were sufficient to enable these proteins to be both effective physiological cysteine protease inhibitors and antibacterial agents.     

胱抑素C早期诊断对比剂肾病的研究

目的 探讨血浆胱抑素C(Cys C)在对比剂肾病(CIN)早期诊断的临床意义.方法 选择使用非离子型低渗造影剂进行冠状动脉造影的260例患者为研究对象,分别于造影前后24,48 h检测Cys C、血清肌酐(Scr)水平,根据改良MDRD公式推算肾小球滤过率(eGFR),根据患者在造影后48 h内是否发生CIN,分为CI...     

冠心病患者冠状动脉病变程度与血清胱抑素C及内皮功能相关性分析

目的：探讨冠心病（CHD）患者冠脉病变程度与血清胱抑素 C（Cys C）及血管内皮功能（RHI）的相关性。方法选取318例接受冠状动脉造影的患者为研究对象,分为对照组（65例）与 CHD 组,根据冠脉病变支数将 CHD 组分单支病变组（77例）、双支病变组（70例）、多支病变组（106例）;根据冠脉病变 Gensini 评分将 CHD 组分为低分组（67例）、中分组（107例）、高分组（79例）;通过外周动脉张力测定（PAT）技术评价血管内皮功能,计算血管反应性充血指数（RHI）;用免疫增强比浊法测定 Cys C 水平。结果随病变支数增加,血清 Cys C 水平增加,RHI 水平减少,组间比较差异有统计学意义（P ＜0．05）,RHI 在双支病变与多支病变组差异无统计学意义（P ＞0．05）;血清 Cys C 随冠脉 Gensini 积分增大而增加（P ＜0．05）,组间比较差异均有统计学意义（P ＜0．05）;血管 RHI 值随冠脉 Gensini 积分增大而减小（P ＜0．05）;且 CHD 患者 Cys C 与冠脉 Gensini 积分程度呈正相关（r＝0．375,P ＜0．01）;RHI 与冠脉 Gensini 积分程度负相关（r＝－0．587,P ＜0．01）;血清 Cys C 与 RHI 负相关（r＝－0．350,P ＜0．01）。结论血管内皮功能障碍、血清 Cys C 的增高与冠脉病变程度相关,且 Cys C 与血管 RHI 呈负相关,血清 Cys C 增高可能是 CHD 患者血管内皮功能受损的预测指标。     

Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a non-inhibitory mode

OBJECTIVES: Our aim was to purify large quantities of human salivary cystatins S, SA and SN in order to determine whether these salivary cystatins have a stable interaction with cysteine proteases at a second binding site, other than the protease active site. This property may affect their availability to act as cysteine protease inhibitors within the oral environment. METHODS: Salivary cystatins S, SA and SN were purified from human submandibular sublingual saliva to homo- geneity by column chromatography. Formation of stable complexes between the model cysteine protease papain in the absence of reductant was assessed by SDS-PAGE and probing Western blots with antibody to human salivary cystatin SN. Proteolytic activity of the complex was determined in the gel after electrophoresis. RESULTS AND CONCLUSIONS: Only cystatin SN (14.3 kD) was found to form a stable complex with papain (22 kD) that could be separated by SDS-PAGE producing a Coomassie stained band at (37 kD). After western transfer this same band (37 kD) cross-reacted with antibody to SN. In the presence of E64, an active site inhibitor of cysteine proteases, the same complex was formed, suggesting that SN is able to bind to papain at a site other than the active site. Activity staining of the gel confirmed that this complex (-E64) retained proteolytic activity. Such complex formation between cystatin SN and cysteine proteases in a non-inhibitory mode may reduce its availability to act as an effective cysteine protease inhibitor in the oral environment.