Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Type 1 diabetes mellitus (T1DM) results from the destruction of beta cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.
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PMID:HLA class I epitope discovery in type 1 diabetes. 1713 May 54

Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.
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PMID:Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons. 2040 34