Lysosomal Cystine Enhanced Apoptosis in Cultured Human Mesenchymal Stem Cells
12 Month Progress Report to the Cystinosis Research Foundation:
Lysosomal Cystine Enhanced Apoptosis in Cultured Human Mesenchymal Stem Cells
Period: 1 Dec 06 - 30 Nov 07
Jess Thoene, MD
University of Michigan
Based on our earlier published work, we hypothesize that proteins in the apoptotic cascade will be cysteinylated by lysosomal cystine during the early stages of apoptosis, thus increasing the rate of apoptosis in cystinotic cells.We have demonstrated for PKC d that such modification can increase enzymatic activity, thus enhancing the aoptotic cascade, leading to inappropriate cell death, and hence the cystinotic phenotype.
As described in the original application, we expose cultures of cystinotic and normal cells to an apoptotic stimuls (TNF a), harvest the cells at appropriate intervals, and analyze the cell proteins by MALDI mass spectrometry. We are assisted in this portion of the work by the University of Michigan Proteome Consortium. To identify which proteins have been cysteinylated by released lysosomal cystine, we first block existing thiols at the time of harvest, either with iodoacetamide, or ICAT, a reagent from Applied Biosystems that has a thiol-specific reactive group adjacent to an alkyl linker, which contains either nine [12C] or nine [13C] atoms - thus resulting in a mass difference of 9 daltons between the control versus the corresponding experimental version of the same tryptic peptide. The cell proteins are then reduced with phosphine and labeled with ICAT heavy or light. The resulting protein pool is then digested, ICAT-labelled proteins isolated via an avidin column, and then analyzed by mass spectrometry. A difference in proteins in normal versus cystinotic cells labeled after native thiols are blocked should identify those proteins cysteinylated by lysosomal cystine after an apoptotic stimulus.
At this point we have identified 3 candidate proteins that meet the listed criteria in at least one trial. One of the three, MFGE8 has been identified in two independent experiments. Gratifyingly, all three contain cyste(i)ne residues, and are thought to have some role in apoptosis. Two, calmegin and calreticulin may work together in the apoptotic cascade. A brief synopsis of the three proteins, listing their aminoacid sequence, and a summary of their biologic function is given below:
1) ER Calcium and ER Chaperones: New Players in Apoptosis?
Edited by: Paul Eggleton and Marek Michalak
Chapter authors: ?Nicolas Demaurex, Maud Frieden and Serge Arnaudeau
By using calcium ions as an intracellular messenger, cells walk a tight rope between life and death. Because critical cellular functions depend on the precise delivery of Ca2+ at the right time and place, calcium ions must navigate at all times between intracellular calcium stores and target proteins located in the cytosol, the mitochondria, or the nucleus. Due to the toxicity of high Ca2+ concentrations, even slight disruption of the elaborate calcium signaling machinery can have devastating consequences on cell functions: too much or too little calcium at the wrong time and place might lead to rapid cell death by necrosis, or to the induction of the cell death program of apoptosis. ER chaperones, and most notably calreticulin, play a key role in the making and decoding of both normal and pathological calcium signals. Calreticulin is the main Ca2+-binding protein residing in the ER, and as such contributes most of the ER Ca2+ buffering capacity. Calreticulin also acts as a chaperone for several ER Ca2+ transport proteins, and thus indirectly modulates Ca2+ fluxes across the ER membrane. Accordingly, over- or underexpression of calreticulin leads to rapid and severe alterations in ER Ca2+ homeostasis. Calreticulin expression levels are controlled by the ER Ca2+ levels, thus enabling cells to mount an appropriate response during long-term perturbations in ER Ca2+ storage. However, calreticulin levels are also increased by a variety of cellular stress conditions, and this upregulation might contribute to the Ca2+ signaling defects leading to apoptosis. In this chapter, we will review the role of calreticulin and of other ER chaperones in the control of Ca2+-mediated apoptosis.
Sequence of Calreticulin: 3 cys residues
1 mllsvplllg llglavaepa vyfkeqfldg dgwtsrwies khksdfgkfv lssgkfygde
61 ekdkglqtsq darfyalsas fepfsnkgqt lvvqftvkhe qnidcgggyv klfpnsldqt
121 dmhgdseyni mfgpdicgpg tkkvhvifny kgknvlinkd irckddefth lytlivrpdn
181 tyevkidnsq vesgsleddw dflppkkikd pdaskpedwd erakiddptd skpedwdkpe
241 hipdpdakkp edwdeemdge weppviqnpe ykgewkprqi dnpdykgtwi hpeidnpeys
301 pdpsiyaydn fgvlgldlwq vksgtifdnf litndeayae efgnetwgvt kaaekqmkdk
361 qdeeqrlkee eedkkrkeee eaedkedded kdedeedeed keedeeedvp gqakdel
1) Molecular Chaperone Calmegin Localization to the Endoplasmic Reticulum of Meiotic and Post-meiotic Germ Cells in the Mouse Testis
Kazuya YOSHINAGA1), Ichiro TANII1) and Kiyotaka TOSHIMORI1)
Archives of Histology and Cytology
Vol. 62 (1999) , No. 3 p.283-293
Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College
Summary. Calmegin is a testis-specific Ca2+-binding protein that is homologous to calnexin. Recently, sperm from transgenic mice lacking calmegin have been shown to be infertile. To further characterize calmegin, we analyzed the precise stage of expression and the intracellular localization of this protein in germ cells during mouse spermatogenesis by an immunoperoxidase technique using the anti-calmegin monoclonal antibody TRA369. Light microscopic immunocytochemistry showed that calmegin appeared in early pachytene spermatocytes, with the highest expression in round and elongating spermatids, and disappeared in the maturation phase of spematids at step 15. Immunoelectron microscopy showed that selective localization was found at the endoplasmic reticulum membrane and the nuclear envelope of spermatogenic cells. During the maturation phase, a dramatic reduction in calmegin occurred in the endoplasmic reticulum of the spermatids, suggesting that the major function of calmegin has been completed by the time spermatids reach step 14. In addition, although the immunoreactivity was completely absent in the calmegin-deficient mutant mouse testis, ultrastructural analysis showed that mature sperm from the knockout mice were normal. This suggests that calmegin is not required for the morphogenesis of male germ cells. Thus, our results suggest that calmegin has a major role in mouse spermatogenesis, and also indicate that this protein would be useful as a maker molecule to study the functional role of the endoplasmic reticulum in the process of spermatid differentiation.
Sequence of human calmegin: 8 cys residues
1 mhfqafwlcl gllfisinae fmdddveted feenseeidv neselsseik yktpqpigev
61 yfaetfdsgr lagwvlskak kddmdeeisi ydgrweieel kenqvpgdrg lvlksrakhh
121 aisavlakpf ifadkplivq yevnfqdgid cggayiklla dtddlilenf ydktsyiimf
181 gpdkcgedyk lhfifrhkhp ktgvfeekha kppdvdlkkf ftdrkthlyt lvmnpddtfe
241 vlvdqtvvnk gslledvvpp ikppkeiedp ndkkpeewde rakipdpsav kpedwdesep
301 aqiedssvvk pagwlddepk fipdpnaekp ddwnedtdge weapqilnpa crigcgewkp
361 pmidnpkykg vwrpplvdnp nyqgiwsprk ipnpdyfedd hpflltsfsa lglelwsmts
421 diyfdnfiic sekevadhwa adgwrwkimi anankpgvlk qlmaaaeghp wlwliylvta
481 gvpialitsf cwprkvkkkh kdteykktdi cipqtkgvle qeekeekaal ekpmdleeek
541 kqndgemlek eeesepeeks eeeieiiegq eesnqsnksg sedemkeadestgsgdgpik
1)Identification of a factor that links apoptotic cells to phagocytes.Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Nature. 2002 May 9;417(6885):182-7 Department of Genetics, University Medical School, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan.
Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif--it bound particularly strongly to cells expressing alpha(v)beta(3) integrin. The NIH3T3 cell transformants that expressed a high level of alpha(v)beta(3) integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.
MFGE8 Protein sequence : 14 cys residues
1 mprprllaal cgallcapsl lvaldicskn pchngglcee isqevrgdvf psytctclkg
61 yagnhcetkc veplgmengn iansqiaass vrvtflglqh wvpelarlnr agmvnawtps
121 snddnpwiqv nllrrmwvtg vvtqgasrla sheylkafkv ayslnghefd fihdvnkkhk
181 efvgnwnkna vhvnlfetpv eaqyvrlypt schtactlrf ellgcelngc anplglknns
241 ipdkqitass syktwglhlf swnpsyarld kqgnfnawva gsygndqwlq ifpgnwdnhs
301 hkknlfetpi laryvrilpv awhnrialrl ellgc
The method seems to effectively identify candidate proteins cysteinylated only in cystinotic cells after an apoptotic stimulus. The three proteins identified so far posses cystine residues, thus are able to be cysteinylated, and they are involved, at least to some extent, in apoptosis. Calreticulin is involved in spermatogenesis. We speculate that abnormal activity of calreticulin in the testes of adolescent cystinotic males could be an explanation for the male infertility seen in this disease.
The major concern we have had is the lack of sensitivity of MALDI to enable detection of the scarce, but powerful regulatory proteins involved in the apoptotic cascade.
We plan to address this issue in two ways:
1) Increase the number of cells harvested at each point: Previously we have harvested about 10*6 cells per condition and time point. We will increase this by a factor of 5 in the next series.
2) Alter the method of harvesting: We have used scraping in order to avoid partial digestion of proteins at harvest which would cloud the MALDI results. However, it is possible that scraping is lysing a substantial proportion of cells, thus releasing the cystosolic proteins into the harvest buffer , where they are lost to analysis. We will compare the identity of proteins obtained from cells harvested by trypsinization with that obtained by scraping in order to determine if this will enhance the yield of identifiable proteins.