Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain 4.2.5.2 Localization of CD38 on Percoll purified mitochondria using Scanning Electron Microscopy (SEM). The results obtained in the localization study of CD38 on mitochondria extracted from mouse brain tissues, labeled with 15nm colloidal gold marker, after mild prefixation, are illustrated in Figure 4.21-4.23. CD38 labeling was observed in the BEI (Backscatter electron imaging) mode. The contrast is dependent on the atomic number, and the gold marker (Au; Z=79) is rendered in high contrast, as well as presenting a clearly distinguishable matrix structure of mitochondria. The gold particles were well visualized in the BEI mode because the 20kV electron beam penetrates most surface structures and good contrast. It is well established that cell surface labeling with SEM is useful as direct correlation between the presence of specific molecules exposed on cell surfaces (antigen, receptor sites, etc) and the surface structure of these same cells (de Harven et al., 1984). Thus the comparison of the SEM (Figures 4.21-4.23) and TEM (Figure 4.18) with gold labeling of CD38 on mitochondria showed that the localization of the molecule is restricted to the cell surface. It is noted that the number of gold particles per surface area is markedly higher in the WT mouse brain mitochondria as compare to the KO mouse brain mitochondria (Figure 4.22). This is interpreted as reflecting the high specificity of the CD38 antibody used which showed negligible background or non-specific staining (Figure 4.22 & 4.23). The inset shows the enlargement of the encircled area, B, of the isolated WT mouse brain mitochondria (Figure 4.22 B) as well as the isolated CD38 KO mouse brain mitochondria (Figure 4.23 A-C). The encircled areas in Figure 4.22, A-F were observed with intense distribution of gold particles which appeared white in BEI mode. In contrast, CD38 KO mouse brain mitochondria in Figure 4.23 shows 199 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain negative staining, as indicated by negligible observation of gold particles as compared to the WT. A closer look at the samples by higher magnification (Figure 4.23 A-C) supported the observations of gold particle staining on the WT sample. There was negligible staining for the CD38 KO samples. The same portion of the same sample was illustrated by superimposition of both the BEI and the SEI (Secondary Electron Imaging) signals (signal mixing (Becker et al., 1979). The polarity of the backscatter signal was set back to ‘normal’ and the SEI and BEI signals were overlaid. The result was an image in which the distribution of all the gold particles can be precisely correlated with details of the surface structure of the sample. It was observed that the majority of discernible gold markers were associated on the surface of the purified intact mitochondria (Figure 4.21, 4.22). Collectively, the present data further supported the outer mitochondrial location of CD38 and strongly agreed to the specific topology with protrusion of Cterminal domain outside the mitochondria, which has consistently observed in the earlier experiments (Chapter 3), as well as the TEM, digitonin titration and protease protection assay. 200 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain B A C D F B E B Figure 4.22 Backscatter electron imaging of mitochondrial fractions, labeled with goat polyclonal CD38 antibody-sc-7049. Backscatter electron micrograph at low magnification with the labeling of CD38 on purified mitochondria isolated form WT mice brain tissues. The encircled areas (A-F) are CD38 immunoreactive region that detected by conjugated gold particles. The inset is enlargement of the encircled area (B) of the isolated mitochondria. Scale bar: 1µm 201 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B C Figure 4.23 Backscatter electron imaging of CD38 KO mitochondrial fractions, labeled with goat polyclonal CD38 antibody-sc-7049. Backscatter electron micrograph at low magnification with the labeling of CD38 on purified mitochondria isolated form CD38KO mice brain tissues. The encircled areas (A-C, please refer to next page) were examined at higher magnification for positive staining of CD38. The insets show enlargement of the encircled area (A-C shown in the following page) with negligible CD38 staining. Scale bar: 1µm 202 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain A B C 203 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain 4.3 Discussion Although the possible presence of functional CD38 in the mitochondria was previously shown using specific organelle targeting CD38 expression system in cell lines (Chapter 3), this is the first conclusive evidence of the presence of CD38, a hitherto characterized multifunctional enzyme, on the mitochondria membrane via an endogenous system, namely highly purified mitochondria isolated from mouse brain tissues. In addition to the finding of intracellular CD38 localized on nucleus, ER (Adebanjo et al., 1999; Khoo et al., 2000; Sun et al., 2002), the results described in this study provide an alternative resolution to the topological issue of this molecule. Studies on NAD+ glycohydrolase activities in mitochondria started back in the 1980s. Moser et al. (1983), Masmoudi et al. (1987;1988) and Hilz et al. (1984) have independently reported an unidentified NAD+ glycohydrolase activities in mitochondria isolated from rat liver, brain and heart tissues, respectively. All known observations of mitochondria NAD+ glycohydrolase showed high activity with NAD+, poly-(ADP-ribose) polymerase activity was not detected in the specific enzyme and it was susceptible to inhibition by nicotinamide and ATP (Moser et al., 1983; Masmoudi et al., 1988). The fact that this NAD+ glycohydrolase observed in the brain mitochondrial preparation was identified as likely to be CD38 in the present study (Table 4.1) suggests that the subcellular distribution of this molecule is more complex than originally thought. More information about the mitochondrial NAD+ glycohydrolase was revealed in the next two decades. Ziegler et al. (1997; 1997b) and Liang et al. (1999) reported independently of mitochondrial NAD+ glycohydrolase activity observed in bovine liver and rat liver tissues. Both Ziegler and Liang groups’ investigations identified the liver mitochondrial NAD+ glycohydrolase as a member of the class of bifunctional 204 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, which is involved in synthesis and degradation of cADPR, the well characterized potent intracellular calciummobilizing agent. NAADP was first shown produced in rat liver mitochondrial NAD+ glycohydrolase by Liang et al. (1999). Ziegler group reported that the bovine mitochondrial NAD+ glycohydrolase showed comparable yield of cADPR and ADPR to CD38. It was also found that Zn2+ increased the yield of cADPR by more than fold in bovine NAD+ glycohydrolase (Zocchi et al., 1993; Ziegler et al., 1997). This result was contradicted by Liang group which showed otherwise. The properties of NAD+ glycohydrolase from a given tissue may vary widely with the species and, within a species, may vary from tissue to tissue (Green et al., 1964). This may account for the discrepancy. Interestingly, Liang group detected a minimal quantity of CD38-like antigen in liver mitochondria. Lisa et al. (2001) reported that 92% of NAD+ glycohydrolase activity in rat heart was found in mitochondria. Aksoy et al. (2006) has provided substantial evidence that gave support to the novel concept of CD38, a widely recognized NAD+ glycohydrolase as the major regulator of intracellular NAD+ in several tissues including brain. In agreement to this, the total mitochondria fraction isolated from mouse brain tissues in the present study showed significantly higher NAD+ glycohydrolase activity as compared to ADP-ribosyl cyclase activity (Chapter Section 4.2.4.2). No apparent cyclase and NAD+ glycohydrolase activities were observed in CD38KO mouse brain mitochondria samples (Figure 4.9), which is in agreement with the finding reported in Aksoy et al., 2006. Hence, the present data demonstrating presence of CD38 on mitochondria is well supported and eliminates the possibility of contamination by unidentified ADP-ribosyl cyclase (Ceni et al., 2003b; 2006) known to be found in CD38KO mouse brain tissues. 205 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain To learn about the cellular function of this mitochondrial CD38, it is important to examine its precise localization on the organelle. Mitochondria are compartmentalized into matrix, inner mitochondria membrane, intermembrane space and outer mitochondrial membrane, as discussed in Chapter 3. The outer membrane has different characteristics including different cholesterol composition as well as different protein composition from the inner mitochondrial membrane. Both membranes come into close contact showing membrane contact points, which are important in regulation of the mitochondria-cytoplasmic exchanges of metabolites, proteins, and phospholipids (Brdiczka, 1991). Recent data argues that mitochondrial NAD+ glycohydrolase is localized to the outer mitochondrial membrane (Boyer et al., 1993; Yamada et al., 1997; Lisa et al., 2001) in contrast to the previous believes that mitochondrial NAD+ glycohydrolase is localized to the inner side of the inner mitochondrial membrane (Lotscher et al., 1980; Moser et al., 1983). It was well established that a loss of intramitochondrial pyridine nucleotides occurs during prooxidant mediated Ca2+ efflux from Ca2+ loaded mitochondrial. While the inner membrane of mammalian mitochondria is normally impermeable to pyridine nucleotides, it was shown that the pyridine nucleotides could be transported through the inner mitochondrial membrane via the opening of the permeability transition pore (PTP) (Boyer et al., 1993; Lisa et al., 2001). Lisa et al further showed that the redistribution of NAD+ between these compartments is made possible by PTP opening because mitochondrial swelling precedes NAD+ hydrolysis mediated by NAD+ glycohydrolase. Thus the pyridine nucleotides are transported to the outer mitochondrial membrane where hydrolysis takes place. 206 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain In agreement with the published data mentioned above, the results obtained from digitonin titration showed a complete solubilisation of CD38 molecule at ≤ 0.1mg/mg of digitonin to protein ratio, indicated that the mitochondrial location of CD38 co-localizes with Tom20 as well as Bcl-xL (the known outer mitochondrial membrane markers) suggesting that its location is the outer mitochondrial membrane (Figure 4.7). This is further supported by the integrity of the intermembrane space proteins and inner mitochondrial membrane proteins under the same treatment. More evidence gathered when protease protection assay was introduced into the digitonin solubilization system offered further confirmation of the location of CD38 to the outer mitochondrial as well as the specific topology in which its carboxyl catalytic domain extruding to the cytosolic region. These pieces of evidence include 1) CD38, as well as the outer mitochondrial membrane markers, Tom20 and Bcl-xL were susceptible to protease digestion even before subjected to digitonin solubilisation; 2) The CD38 antibody, sc7049 (M19, Santa Cruz) employed here detects epitope mapped to extracellular domain of the molecule. However, the nature of the study was unable to reach definitive conclusions with regards to the precise topography of CD38 on the outer mitochondrial membrane. This is due to the limitation of choices with immunoreactive mouse CD38 antibody. The protein topology can be further investigated by means of peptide antibodies raised against the carboxyl-terminal region of the protein. Endoplasmic reticulum/microsomal vesicles have been shown to contain CD38 molecule (Sun et al., 2002), and mitochondrial preparation are commonly contaminated with varying amounts of microsomes (Schnaitman et al., 1968). From the data presented (Figure 4.6), isolated purified mitochondria was shown to be devoid of ER as well as plasma membrane markers. Thus the results of this study 207 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain indicate that CD38 observed in Percoll purified mouse brain mitochondrial preparations is a native constituent of the outer mitochondrial membrane and that the outer membrane is the only mitochondrial location for this molecule. The results obtained from digitonin titration and protease protection assay were further verified by employing electron microscopic techniques. The localization of CD38 as well as the topography on the mitochondrial was examined by combining TEM and SEM methods. The TEM consistently showed that the distribution of DAB staining was strictly localized to the outer rims of mitochondria, as would be expected for protein associated in the outer mitochondrial membrane. This is generally in agreement with those reported by Yamada et al. (1997). Three CD38 antibodies were employed in the immunostaining experiment, two polyclonal CD38 commercialised antibodies and a lab customized peptide anti-human CD38 antibody. All three antibodies recognized CD38 and showed the same staining pattern. This is not surprising as it is well established that murine CD38 cDNA shows 70% sequence homology with that of human CD38 (Harada et al., 1993). Consistent labelling of intracellular CD38 on nuclear envelope was not observed; in fact the intracellular CD38 staining was highly heterogeneous but remained constant for plasma membrane and postsynaptic densities. The difference could be due to the use of mouse brain tissues in current study as opposed to use of rat brain tissues in Yamada’s data. It is noteworthy that the staining pattern on mitochondria is specific and heterogeneous; uneven rather than uniform distribution of CD38 associated with the outer mitochondrial membrane was observed. Not all mitochondria in the immunopositive cells were stained and not all cells are immunoreactive to CD38 (Figure 4.10-4.17). The observation is interesting, in account of accumulative research in the connectivity and functional homogeneity of mitochondria in cells; recent publications showed the 208 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain dynamic nature of mitochondria within individual cells, including neurons, which could be morphologically heterogeneous and unconnected thus allowing them to have distinct functional properties (Collins et al., 2002). This may suggest that individual mitochondria could have a specific role in response to the local environment/ consequence to factors that can dynamically regulate functional aspects of mitochondria such as ∆ψmit and Ca2+ sequestration (Jouaville et al., 1995; 1999). It is possible that the heterogeneous expression of CD38 observed on each individual mitochondrion may have a role to its respective functions. The mild fixation conditions and the optimized tonicity of the media used in the DAB procedure would tend to argue against any artifactual rearrangement of the mitochondrial membranes during experimental manipulation. All of the above considerations strongly suggest that the DAB stain accurately reflects the in vivo configuration of the mitochondrial membranes and not giving artifactual CD38 staining. A more detailed observation of the topographic structure of CD38 was obtained using high-resolution investigation with ISEM. The data collected implied that mitochondrial CD38 is associated with the surface of mitochondria. Specific CD38 staining techniques making use of simultaneous detection of back-scatter electrons imaging (BEI) and secondary electrons imaging (SEI) have provided valuable information on the distribution of mitochondrial CD38 on the surface of the organelle. This method was first attempted for the study of the specific localization of cell surface antigenic sites labelled with particles of colloidal gold. It was illustrated by its application to the identification of human circulating granulocytes (De Harven et al., 1984). It was advantageous to view the sample using BEI as the high atomic number of gold particles gives good contrast on the surface carbon-coated 209 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain mitochondria samples (De Harven et al., 1984), so that this can be used as a complementary technique to SEI to provide unequivocal identification of all gold markers on the surface architecture of the samples. Not all mitochondria samples displayed precise cristae structure under BEI mode; this variation may be due to the difference in focal length relative to the varying distance of the detector, which was in a fixed position, to the specimen as well as particular structural features in different positions within the specimen (Wells, 1977). By superimposing the SEI and BEI signals, a 3D conformation that confirmed the localization of CD38 antigen with its catalytic site protruding out from the outer mitochondrial membrane was obtained without losing the image of the accompanying organelles’ surface structure. There are limitations of this system such as the tendency of isolated mitochondria to form clumps and therefore obscuring the topographical visualization of mitochondrial CD38. Also the low endogenous expression of mitochondrial CD38 and limitations to the choices of effective mouse CD38 antibodies remain as challenges which need to be addressed in future studies. The results that obtained from immunoelectron microscopy and stepwise digitonin titration method combining protease protection assay agreed with its localization to the outer mitochondrial membrane and further suggests that the extracellular domain of mitochondrial CD38 is facing the cytosolic side. This was unexpected as the fact that CD38 is transported to the cell membrane surface with its extracellular domain (carboxyl-terminal domain) containing the enzymatic site facing the extracellular region would suggest luminal localization of the CD38 enzymatic site for intracellular CD38. Indeed, up to date observations of intracellular CD38 show that it is normally found facing the lumen/intravesicular area (Sun et al., 2002, Davis et al., 2008). This yet to be explained topological heterogeneity is intriguing 210 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain and surprising. Indeed, possible explanations was postulated in the recent study that polypeptides with identical sequences can span the membrane differently (Ott and Lingappa. 2002). Nevertheless, the present data strongly argue against cross contamination of CD38 from other cellular compartment. The question of the possible role of CD38 localized on the mitochondria is therefore fascinating. So the next question would be could CD38 found in mitochondria with such specific and unusual topology have a role in the calcium signaling involving second messengers such as cADPR, NAADP and ADPR? The role of mitochondrial CD38 in calcium signalling was investigated and reported in Chapter 3. Sun et al. (2002) reported that full cytosolic Ca2+ responses can be triggered by CD38 (including intracellular expressed CD38 in ER, mitochondria) with its enzymatic site facing the cytosol. In agreement with this, the results obtained in this present study indicated that mitochondria localized CD38 molecules are able to initiate Ca2+ release mechanism (Chapter section 3.2.6). Taken together, these results lead to a proposition that mitochondrial CD38 localised on outer mitochondrial membrane with the extracellular catalytic site facing the cytosol is expected to have a convenient role in the synthesis of cADPR as well as ADPR and thus involved in intracellular Ca2+ signalling. In the present study, the release of Ca2+ into the cytosol was investigated using a simulated in vivo system. The system involved isolated mitochondria expressing Mito-CD38 together with isolated Ca2+ loaded ER. Ca2+ release was monitored using Fluo-3. The system was established to allow the synthesis and action of cADPR to trigger the release of Ca2+ from ER Ca2+ store via RYR. When incubated together, CD38+ localized on the intact mitochondria responded to β-NAD+ which in turn produced cADPR resulting in the triggered 211 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain [Ca2+] release from the cADPR-sensitive Ca2+ store. The result was further supported by the addition of cADPR antagonist, 8-Bromo-cADPR which inhibited the Ca2+ release completely. On the other hand, it is interesting to note that in neurons, the ryanodinesensitive stores have thus far been found mainly localized in cell soma, whereas the IP3-sensitive stores appear to be equally distributed in neuronal soma and processes (Thayer et al., 1986; 1988; Martinez-Serrano et al., 1989). Interestingly, ubiquitous CD38+mitochondria were observed in both neuronal soma and processes in the ultrastructural localization studies of CD38 in mouse brain tissues using TEM (Figure 4.10-4.17). Also, it has been noticed in the present study that CD38+mitochondria are in close proximity with the ER as well as plasma membrane (Figure 4.10, 4.14). Close appositions between ER (or sarcoplasmic reticulum, SR) and mitochondria have long been known to exist and have been observed by electron microscopy (EM) in several cell types. These regions have been known to constitute sites of phospholipid exchange between the two organelles which may implicate trafficking between two organelles (refer to Chapter for more detailed discussion on ERMitochondria trafficking). Taking all the information together, it is very tempting to hypothesize with such close proximity between mitochondria and ER, cADPR synthesized by mitochondrial CD38 would then ‘conveniently’ acts on the RYR which localized on ER and triggers the immediate release of ER Ca2+ store. However, it is necessary to redefine and “re-explore” the Ca2+release assay in the current study, given that it was carried out in vitro and some cytosolic factors that may increase effectiveness in mitochondrial CD38 mediated Ca2+ signaling as well as ER Ca2+-store Ca2+ affinity 212 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain might be compromised during organelle isolation. For example, cytosolic factors like calmodulin, which has been shown by Lee and co-workers to be required and needs to be present for the full activity of cADPR (Lee et al., 1994). Re-establishing an in vivo Ca2+ signaling system is therefore essential to allow further characterization the roles of CD38 found in mitochondria. Recently, ryanodine receptor has been reported to be localized in the inner membrane of mitochondria (Beutner et al., 2001), which opened the possibility that cADPR may act on mitochondrial Ca2+ homeostasis. Therefore it would be interesting to investigate the possible relationship between the mitochondrial CD38 and ryanodine receptor. A substantial amount of cellular NAD+ pool i.e, ~75% of intracellular NAD+ in heart muscle, is compartmentalized within the mitochondria’s matrix (Tischler et al., 1977; Di Lisa et al., 2001). It should be duly noted that NAD+ is also present in the cytosol. The question is how does matrix storage of NAD+ reach the CD38/NAD+ glycohydrolase which are localized on the outer mitochondrial membrane given that inner mitochondrial membrane is not permeabilised to solutes? Di Lisa group provided evidence using post-ischemic reperfusion injury of heart tissues, and showed that mitochondria respond to the assault by opening the PTP which caused the release of NAD+ from the matrix store and concluded that NAD+ hydrolysis was only made possible after the PTP opening. With the extracellular catalytic domain extruding out into the cytosol, this could serve as a regulatory mechanism to control the cADPR production by mitochondrial CD38 and thus Ca2+ release signaling, given that this outer mitochondrial membrane localized NAD+ glycohydrolase is shielded from the matrix NAD+ store. No doubt cytosolic NAD+ can be contributed from nucleus NAD+ storage so how would this molecule with it catalytic domain exposed in the cytosolic 213 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain region be regulated? It is then believed that membrane glycohydrolase works more efficiently in the vicinity of substrate production site together with the immediate availability of substrate. In this case, it would be the ‘immediate’ NAD+ ‘supply’ from the matrix storage which will only be available when the PTP open. Previous studies has shown that the NAD+ glycohydrolase in the nuclear envelope is present in a latent form and can be activated when triggered by stimuli (Tamulevicius et al., 1979). Another study by Sato et al. (1999) revealed a novel peptide inhibitor to human BST-1/CD157. This cADPR-synthesizing enzyme and CD38 are believed to have evolved from a common ancestor by gene duplication (Ferrero and Malavasi, 1999). Taken together, it is therefore not inconceivable that there exist inhibitor peptides or proteins to regulate CD38 activity intracellularly. Grimaldi et al., (1995) have shown that CD38 is able to ADP-ribosylate certain proteins in vitro via the cysteine moiety in a non-enzymatic manner. It has long been reported that NAD+ glycohydrolase as well as ADP-ribosylation activities are present in heart, liver and brain mitochondria (Moser et al., 1983; Masmoudi et al., 1986; 1988; Boyer et al., 1993; Ziegler et al., 1997; 1997b; Lisa et al., 2001). It is possible that mitochondrial CD38 can function as NAD+ glycohydrolase as well as have a role in ADP-ribosylating specific proteins in the mitochondria to accomplish post translational modification of these proteins or it may control the level of ADPribosylation by limiting the substrate availability for ADP-ribosyltransferase available in close proximity (Krebs et al., 2005). It was postulated that such ADP-ribosylated proteins may have an impact on Ca2+ signaling in mitochondria (Ziegler, 2000). Additionally, Grimaldi et al., (1995) also showed that CD38 can undergo autoribosylation and Han et al., (2000) reported that ADP-ribosylation inhibits the 214 Chapter Characterization of CD38 Expressed in Mitochondia from Murine Brain enzymatic activities of CD38. Together, the information point to possible regulation on mitochondrial CD38. Increasing attention has been placed on the role of intracellular CD38 in the recent years. The possibility that expressed intracellular CD38 has an important role in intracellular signaling and yet maintaining its membrane bound locality is important and cannot be ignored. In the present study, for the first time, the localization of enzymatically active CD38 to the outer mitochondrial membrane of mouse brain tissues is strongly suggested and shown with specific topology that the carboxyl catalytic domain of the molecule is facing the cytosolic side. 215 [...]... argue against any artifactual rearrangement of the mitochondrial membranes during experimental manipulation All of the above considerations strongly suggest that the DAB stain accurately reflects the in vivo configuration of the mitochondrial membranes and not giving artifactual CD38 staining A more detailed observation of the topographic structure of CD38 was obtained using high-resolution investigation... mitochondrial CD38 localised on outer mitochondrial membrane with the extracellular catalytic site facing the cytosol is expected to have a convenient role in the synthesis of cADPR as well as ADPR and thus involved in intracellular Ca2+ signalling In the present study, the release of Ca2+ into the cytosol was investigated using a simulated in vivo system The system involved isolated mitochondria expressing... Ca2+release assay in the current study, given that it was carried out in vitro and some cytosolic factors that may increase effectiveness in mitochondrial CD38 mediated Ca2+ signaling as well as ER Ca2+-store Ca2+ affinity 212 Chapter 4 Characterization of CD38 Expressed in Mitochondia from Murine Brain might be compromised during organelle isolation For example, cytosolic factors like calmodulin, which has... signaling in mitochondria (Ziegler, 2000) Additionally, Grimaldi et al., (1995) also showed that CD38 can undergo autoribosylation and Han et al., (2000) reported that ADP-ribosylation inhibits the 214 Chapter 4 Characterization of CD38 Expressed in Mitochondia from Murine Brain enzymatic activities of CD38 Together, the information point to possible regulation on mitochondrial CD38 Increasing attention has... glycohydrolase as well as have a role in ADP-ribosylating specific proteins in the mitochondria to accomplish post translational modification of these proteins or it may control the level of ADPribosylation by limiting the substrate availability for ADP-ribosyltransferase available in close proximity (Krebs et al., 2005) It was postulated that such ADP-ribosylated proteins may have an impact on Ca2+ signaling... attention has been placed on the role of intracellular CD38 in the recent years The possibility that expressed intracellular CD38 has an important role in intracellular signaling and yet maintaining its membrane bound locality is important and cannot be ignored In the present study, for the first time, the localization of enzymatically active CD38 to the outer mitochondrial membrane of mouse brain tissues is... role of CD38 localized on the mitochondria is therefore fascinating So the next question would be could CD38 found in mitochondria with such specific and unusual topology have a role in the calcium signaling involving second messengers such as cADPR, NAADP and ADPR? The role of mitochondrial CD38 in calcium signalling was investigated and reported in Chapter 3 Sun et al (2002) reported that full cytosolic... domain (carboxyl-terminal domain) containing the enzymatic site facing the extracellular region would suggest luminal localization of the CD38 enzymatic site for intracellular CD38 Indeed, up to date observations of intracellular CD38 show that it is normally found facing the lumen/intravesicular area (Sun et al., 2002, Davis et al., 2008) This yet to be explained topological heterogeneity is intriguing... that cADPR may act on mitochondrial Ca2+ homeostasis Therefore it would be interesting to investigate the possible relationship between the mitochondrial CD38 and ryanodine receptor A substantial amount of cellular NAD+ pool i.e, ~75 % of intracellular NAD+ in heart muscle, is compartmentalized within the mitochondria s matrix (Tischler et al., 1 977 ; Di Lisa et al., 2001) It should be duly noted that... that obtained from immunoelectron microscopy and stepwise digitonin titration method combining protease protection assay agreed with its localization to the outer mitochondrial membrane and further suggests that the extracellular domain of mitochondrial CD38 is facing the cytosolic side This was unexpected as the fact that CD38 is transported to the cell membrane surface with its extracellular domain . synthesis of cADPR as well as ADPR and thus involved in intracellular Ca 2+ signalling. In the present study, the release of Ca 2+ into the cytosol was investigated using a simulated in vivo. that it was carried out in vitro and some cytosolic factors that may increase effectiveness in mitochondrial CD38 mediated Ca 2+ signaling as well as ER Ca 2+ -store Ca 2+ affinity Chapter. intracellular CD38 in the recent years. The possibility that expressed intracellular CD38 has an important role in intracellular signaling and yet maintaining its membrane bound locality is important and