锰对PC12细胞增殖及蛋白酶体活性的抑制作用

目的：探讨锰对PC12细胞蛋白酶体20s复合物活性的影响.方法：以MTT以及平板克隆形成实验筛选作用浓度,不同时间以及不同浓度作用之后,提取细胞总蛋白,分别检测总蛋白提取液中20s复合物3种酶的活性.结果：含有MnCl2 300μmol/L的培养基对蛋白酶体3种酶活性的抑制率随着作用时间的延长而增高.作用3 d后,对3种酶活性的抑制率均超过20%.在作用时间相同的条件下,随着作用浓度的增高,锰对PC12细胞蛋白酶体活性的抑制作用逐渐增强.同样作用3 d后,当MnCl2浓度达到500μmol/L时,锰对3种酶活性的抑制率均超过40%.结论：蛋白酶体20s活性的抑制可能在锰所诱导的神经毒性过程中发挥着一定的作用.     

KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Because of a high rate of immunoglobulin synthesis, the endoplasmic reticulum of MM cells is subjected to elevated basal levels of stress. Consequently, proteasome inhibitors, which exacerbate this stress by inhibiting ubiquitin-proteasome-mediated protein degradation, are an important new class of chemotherapeutic agents being used to combat this disease. However, MM cells still develop resistance to proteasome inhibitors such as carfilzomib. Toward this end, we have established carfilzomib-resistant derivatives of MM cell lines. We found that resistance to carfilzomib was associated with elevated levels of prosurvival autophagy, and Kruppel-like factor 4 (KLF4) was identified as a contributing factor. Expression levels as well as nuclear localization of KLF4 protein were elevated in MM cells with acquired carfilzomib resistance. Chromatin immunoprecipitations indicated that endogenous KLF4 bound to the promoter regions of the SQSTM1 gene encoding the ubiquitin-binding adaptor protein sequestosome/p62 that links the proteasomal and autophagic protein degradation pathways. Ectopic expression of KLF4 induced upregulation of SQSTM1. On the other hand, inhibitors of autophagy sensitized MM cells to carfilzomib, even in carfilzomib-resistant derivatives having increased expression of the multidrug resistance protein P-glycoprotein. Thus, we report here a novel function for KLF4, one of the Yamanaka reprogramming factors, as being a contributor to autophagy gene expression which moderates preclinical proteasome inhibitor efficacy in MM.     

Proteasome inhibition in hematologic malignancies

Hematologic malignancies, including multiple myeloma (MM), will account for more than 100,000 new cases of cancer and over 57,000 deaths in the United States in 2003. Treatment of MM is a serious challenge, because despite a variety of available therapies, median survival is short. A new therapeutic area focuses on inhibiting the activity of the proteasome, a 26S protease complex involved in cell cycle regulation, cell adhesion, inflammation, and protein turnover. The novel proteasome inhibitor, bortezomib (Velcade®), was recently approved for use in patients with refractory and relapsed MM and to date is the only proteasome inhibitor to have entered clinical trials. Bortezomib has demonstrated activity with manageable toxicity in a variety of hematologic malignancies in addition to MM, including leukemia and non‐Hodgkin's lymphoma. This article reviews clinical information on bortezomib in hematologic malignancies both as monotherapy and in combination with dexamethasone. Preliminary reports of bortezomib in combination with Doxil (pegylated liposomal doxorubicin), melphalan, and thalidomide are discussed, and current trials are described. Available data suggest that bortezomib will be useful in the treatment of a variety of hematologic malignancies.     

Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells

Inhibition of the proteasome has emerged as a clinically effective anticancer therapeutic approach in recent years. Bortezomib (Velcade®) showed extremely high potency against a wide range of cancer cell lines. Ixazomib (MLN9708-MLN2238), the second-generation proteasome inhibitor, selectivity and potency were similar to that of bortezomib, is currently being investigated in phase I studies. It shows superior antitumor activity in hematologic malignancy, especially multiple myelomas. In this study, for the first time, we evaluated and compared the antiproliferative and apoptotic effects of the novel proteasome inhibitor MLN2238 (the active form of MLN9708) with bortezomib using in vitro chronic myeloid leukemia. Cytotoxic and apoptotic effects of MLN2238 and bortezomib were determined by trypan blue dye exclusion assays, WST-1 cell proliferation assay, increased AnnexinV-PI binding capacity, changes in caspase-3 activity and loss of mitochondrial membrane potential (JC-1). Associated with proteasome pathway NFκB1 and c-myc mRNA expression levels were examined by the qRT-PCR method. We observed that cytotoxic and apoptotic effects on K562 cells were started at 5?μm of MLN2238 and 1?μm of bortezomib after 24 and 48?h. Also, MLN2238 and bortezomib downregulated NFκB1 and c-myc mRNA expression at 24?h. Our result revealed that MLN22238 and bortezomib had significant cytotoxic and apoptotic effects on K562 cells. Here, we first demonstrate in vitro data that support the development of MLN2238, by direct comparison with bortezomib on K562 cells.     

The physiological response of protease inhibition in dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by the production of a non‐functional dystrophin gene product and a failure to accumulate functional dystrophin protein in muscle cells. This leads to membrane instability, loss of Ca²⁺ homoeostasis and widespread cellular injury. Associated with these changes are increased protease activities in a variety of proteolytic systems. As such, there have been numerous investigations directed towards determining the therapeutic potential of protease inhibition. In this review, evidence from genetic and/or pharmacological inhibition of proteases as a treatment strategy for DMD is systematically evaluated. Specifically, we review the potential roles of calpain, proteasome, caspase, matrix metalloproteinase and serine protease inhibition as therapeutic approaches for DMD. We conclude that despite early results to the contrary, inhibition of calpain proteases is unlikely to be successful. Conversely, evidence suggests that inhibition of proteasome, matrix metalloproteinases and serine proteases does appear to decrease disease severity. An important caveat to these conclusions, however, is that the fundamental cause of DMD, dystrophin deficiency, is not corrected by this strategy. Hence, this should not be viewed as a cure, but rather, protease inhibitors should be considered for inclusion in a therapeutic cocktail. Physiological Relevance. Selective modulation of protease activity has the potential to profoundly change intracellular physiology resulting in a possible treatment for DMD. However, alteration of protease activities could also lead to worsening of disease progression by promoting the accumulation of substrates in the cell. The balance of benefit and potential damage caused by protease inhibition in human DMD patients is largely unexplored.     

A decade of the anaphase-promoting complex in the nervous system

Control of protein abundance by the ubiquitin–proteasome system is essential for normal brain development and function. Just over a decade ago, the first post-mitotic function of the anaphase-promoting complex, a major cell cycle-regulated E3 ubiquitin ligase, was discovered in the control of axon growth and patterning in the mammalian brain. Since then, a large number of studies have identified additional novel roles for the anaphase-promoting complex in diverse aspects of neuronal connectivity and plasticity in the developing and mature nervous system. In this review, we discuss the functions and mechanisms of the anaphase-promoting complex in neurogenesis, glial differentiation and migration, neuronal survival and metabolism, neuronal morphogenesis, synapse formation and plasticity, and learning and memory. We also provide a perspective on future investigations of the anaphase-promoting complex in neurobiology.     

Opposing effects on immune function and skin barrier regulation by the proteasome inhibitor bortezomib in an allergen‐induced eczema model

Proteasome inhibition (PI) has been reported to interfere with antibody‐driven autoimmune diseases. The impact of PI on the allergic immune response and on skin diseases like atopic dermatitis (AD) has not been thoroughly explored, however. Here, we examined whether the PI bortezomib interferes with the allergic immune response and the severity of AD by using an established mouse model of allergen‐driven dermatitis, to which bortezomib was applied after the establishment of systemic sensitization to ovalbumin. The treatment indeed resulted in a remarkable decrease in total and allergen‐specific plasma cells/antibody‐secreting cells, as evidenced by flow cytometry and ELISpot, respectively. This was accompanied by rapid reductions in serum antibody titres, including a prominent reduction of the IgE isotype. CD4+ and CD8+ cells were greatly diminished in lesional skin on immunohistological staining. The impressive effects at the level of immune modulation did not result in any improvement in the eczema, however. Following up on this unexpected result, we found that the skin itself was susceptible to bortezomib, by which it was instructed to lower the expression of critical skin barrier genes, especially transglutaminase‐1 and filaggrin. Together, bortezomib eliminates plasma cells and decreases immunoglobulin responses, including allergenic IgE. Although anti‐inflammatory effects are detectable in the skin, counter‐regulatory effects from PI on resident skin cells likely undermine improvement in the eczema. These results caution against the therapeutic use of bortezomib for inflammatory skin disorders, which are characterized by inherently impaired barrier function, especially AD.     

Prenatal stress increased Snk Polo-like kinase 2, SCF β-TrCP ubiquitin ligase and ubiquitination of SPAR in the hippocampus of the offspring at adulthood

Exposure to excessive glucocorticoids during fetal development period contributes to later life psychopathology. Prenatal stress decreases dendritic spine density and impair LTP in the hippocampus of rat pups, however, the mechanisms regulating these changes are still unclear.