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Functional Research And Subcellular Localization Of Matrilysin-2

Posted on:2012-08-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z Y WangFull Text:PDF
GTID:1220330368978931Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
MMPs (matrix metalloproteinases) are zinc-dependent peptidases that control the homeostasis of the extracellular matrix. Many extracellular matrix proteins, signal proteins and cytokines are their substrates. MMPs play important functions in many physiological and pathological conditions such as tissue remodeling, arthritis, angiogenesis, tumor metastasis and inflammation. Recent discoveries indicate that MMPs have more complex functions and their location have been considered more important than earlier thought, mislocalization or presentation in unconventional cellular compartments offer MMPs with a chance to degrade new substrates or to perform new functions. Studying the mechanism of cellular distribution or mislocalization of MMPs is critical for understanding their functions.According to their structures, MMPs could be divided into two major groups: secreted and membrane-type MMPs. Recently discovered Matrilysin-2, also known as MMP-26/Endometase, which belongs to secreted MMPs, is partially characterized. The domain structure of MMP-26 is mostly close to MMP-7, as they both have three mini domains including a signal peptide, a pro domain and a catalytic domain. Although they both belong to matrilysin subfamily, few substrates of MMP-26 have been found, most of which are not substrates of MMP-7. Unorthodox autolytic activation mechanism of the MMP-26 zymogen has been identified, which is caused by its non-conservative pro-domain and other atypical structures.Moreover, MMP-7 acted as secreted proteins and showed perinuclear punctate immunocytochemistry stains, and secreted MMP-7 was co-localized with CD44 heparan sulfate proteoglycan on the mammary glands luminal surface, which is its conventional localization. In contrast, studies have demonstrated that MMP-26 expressed mainly in cytoplasm of MCF-7 and ARCaP cells. In vivo experiments indicate that MMP-26 is expressed in cytoplasm and co-localized with laminin-5. Intracellular distribution of MMP-26 is considered as its mislocalization, and a new intracellular substrate estrogen receptor-beta has been found.Intracellular compartmentalization plays an important function in spatial and temporal regulation of many molecular signal processes. While it is established that MMP-26 is mainly localized to the cytoplasm, the exact localization is not known. In order to pinpoint the MMP-26 cellular location and identify the primary sequence and structural basis of MMP-26 intracellular mislocalization, we fused eGFP to the C-terminal of MMP-26, and investigated the distributions of various MMP-26 chimeric eGFP proteins.We first detected the MMP-26 protein expression in HeLa and A549 cells using immuno-cytochemistry and immuno-blot methods with a MMP-26 rabbit polyclonal antibody. Strong positive stains of MMP-26 were both obtained in cytoplasm of HeLa and A549 cells with similar patterns of distribution. Western blot was performed to detect MMP-26 expression in both cells, and proved that MMP-26 was expressed mainly in cytoplasm of HeLa and A549 cells.Subcellular organelles co-localization exparement showed MMP-26WT-eGFP had a strong co-localized stain with ER marker compared with other organelle-markers, which was similar to the endogenous MMP-26. So we concluded that intracellular cytoplasm MMP-26 was specifically localized in ER.As there is no classical ER retention signal in MMP-26 sequence, we considered that its ER retention was caused by a unique mechanism. A signal anchor motif in N-terminal of MMP-23 has been proved for its ER membrane localization. Our results indicated that the N-terminal of MMP-26 functions as a normal signal peptide, and it was similar to MMP-7 signal peptide in guiding secretion of secretory protein, and thus the ER retention of MMP-26 took another different mechanism compared with MMP-23. We constructed two domain-deleted mutants (MMP-26ΔP-eGFP and MMP-26ΔC-eGFP), which localizations showed that Cat domain of MMP-26 has more important role in its ER retention compared with its Pro domain. Next, a serious of C-terminal truncated MMP-26-eGFP fusion proteins had been constructed to identify the relation between its sequence and ER localization. Our results demonstrated that in the presence of the signal peptide, fusion proteins containing N-terminal sequence (80-125) of MMP-26 resided in ER. In contrast, fusion proteins lost portion of this sequence (MMP-261-120-eGFP, MMP261-115-eGFP, MMP-261-135Δ18-90-eGFP, MMP-261-110-eGFP and MMP-261-101-eGFP) failed to reside in ER, indicated that amino acid sequence 80-125 of MMP-26 dictated its ER-retention. Multiple sequence alignment of MMPs indicated that amino acid sequence 80-125 of MMP-26 is composed of a non-conserved region of amino acid in MMPs family. In conclusion, ER retained MMP-26 take a KDEL independent ER retained machenism.As MMP-26 is retained to ER by a KDEL-independent retention signal as shown above, it would be of interest to know whether it can further transported out of the ER by stimuli. DTT, A23187 and Forskolin, which have an obvious effect on ER stress, could induced a perinuclear translocation of ER-localized MMP-26 by immunocytochemistry and Nycodenz density gradient centrifugation analysis. RNAi and RT-PCR showed the amount of vacuolar cells was increased by lower expression of MMP-26, indicated increased cell stress in interfered cells.Endoplasmic reticulum has many functions in cells, one of which is the biggest storage of Ca2+ in cells. MMP-26 needs higher calcium concentrations than that in normal cells, which vary from 300 to 1200 nM, for its correct conformation and activity. The fact that MMP-26 is mostly retained in the ER where the calcium is stored may therefore provide an ideal environment for its folding and activity. The. intracellular ’mis’ location of MMP-26 in ER may instead be the correct location for its physiological function in the cells.
Keywords/Search Tags:ER retained, ER stress, MMP-26, Apoptosis, Ca2+ binding protein
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