Fuyu Yang

Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway

Lysosomes can trigger the mitochondrial apoptotic pathway by releasing proteases. Here we report that a 25-kDa protein purified from rat liver lysosomes possesses a long standing potent Bid cleavage activity at neutral pH, and the truncated Bid can in turn induce rapid mitochondrial release of cytochrome c. This protease was revealed as chymotrypsin B by biochemical and mass spectrometric analysis. Although it was long recognized as a digestive protease exclusively secreted by the exocrine pancreas, our data support that it also expresses and intracellularly resides in rat liver lysosomes. Translocation of lysosomal chymotrypsin B into cytosol was triggered by apoptotic stimuli such as tumor necrosis factor-α, and intracellular delivery of chymotrypsin B protein induced apoptotic cell death with a potency comparable with cathepsin B, suggestive of a lysosomal-mitochondrial pathway to apoptosis regulated by chymotrypsin B following its release. Noteworthily, either knockdown of chymotrypsin B expression by RNA interference or pretreatment with chymotrypsin B inhibitor N-p-tosyl-l-phenylalanine chloromethyl ketone significantly reduced tumor necrosis factor-α-induce apoptosis. These results demonstrate for the first time that chymotrypsin B is not only restricted to the pancreas but can function intracellularly as a pro-apoptotic protease.

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

tBid forms a pore in the liposome membrane

We investigated the ability of tBid (truncated form of Bid) to bind and permeabilize the liposomes (large unilamellar vesicles, LUVs) and release fluorescent marker molecules (fluorescein‐isothiocyanate‐conjugated dextrans, FITC‐dextrans) of various molecular diameters (FD‐20, FD‐70, FD‐250S) from LUVs. Obtained data showed that tBid was more efficient in promoting leakage of FITC‐dextrans from LUVs composed of cardiolipin and dioleoylphosphatidylcholine (DOPC) than LUVs made of dioleoylphosphatidic acid or dioleoylphosphatidylglycerol and DOPC. The leakage efficiency was reduced with increasing amount of dioleoylphosphatidylethanolamine or dielaidoylphosphatidylethanolamine. Phospholipid monolayer assay and fluorescence quenching measurements revealed that tBid inserted deeply into the hydrophobic acyl chain of acidic phospholipids. Taking into account the tBid three‐dimensional structure, we propose that tBid could penetrate into the hydrophobic core of membrane, resulting in the leakage of entrapped content from LUVs via a pore‐forming mechanism.

Lysosomal chymotrypsin B potentiates apoptosis via cleavage of Bid

We have reported that chymotrypsin B (CtrB) is not just a digestive enzyme but is also stored in lysosomes. Herein, we demonstrated a broad distribution of CtrB and explored the involvement of CtrB in apoptosis. Exposure of RH-35 cells to H2O2 or palmitate induced the redistribution of lysosomal CtrB into the cytoplasm as a result of lysosomal membrane permeabilization (LMP). Suppression of CtrB significantly blocked apoptosis, while overexpression of CtrB sensitized apoptosis markedly. CtrB could cleave Bid under neutral conditions. In RH-35 cells with Bid silenced, apoptosis induced by CtrB protein was attenuated, suggesting that CtrB mediates apoptosis of RH-35 cells mainly through processing Bid. Our data also suggest that LMP occurs earlier than mitochondrial outer membrane permeabilization; Bid activation initiated by caspase-8 might be reinforced by CtrB in consequence of LMP, which causes a positive feedback loop leading to the accumulation of tBid, and results in lysosome- and mitochondrion-dependent apoptosis.

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase

The activation and deactivation of Ca2+- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca2+-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca2+ concentrations ([Ca2+]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca2+]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

The Mutation in the N-Terminal Domain of RGS4 Disrupts PA-Conferred Inhibitory Effect on GAP Activity

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins (GAP) for G protein α-subunits and are thought to be responsible for rapid deactivation of G protein mediated signaling pathway. In this present study, we demonstrate that PA is the most efficient candidate to inhibit GAP activity of RGS4. The functional significance of N-terminus of RGS4 in respose to PA-granted inhibition on GAP activity has been studied with the site mutation in the N-terminus of RGS4. These site-directed mutations in the N-terminal domain do not severely disrupt its association with liposomes of PA. However, RGS4L23E diminishes the inhibition of GAP activity by PA compared with the wild type RGS4, whereas RGSR22E abrogates the inhibitory effect by PA on GAP activity. The correspondent conformational discrepancy in the RGS domain of these mutants in the presence of PA vesicles was detected from fluorescence experiments. It is suggested that the functional pertinence between the N-terminus and RGS domain may be important to modulate PA-conferred inhibitory effect on its GAP activity.

Lysosomal chymotrypsin induces mitochondrial fission in apoptotic cells by proteolytic activation of calcineurin

Apoptosis is a fundamental physiological process in mammals in which cells die by activating a suicide mechanism. The mitochondria are one of the major checkpoints in apoptotic regulation because they serve as sensors and amplifiers of cellular damage (Green and Kroemer, 2004). After mitochondrial outer membrane permeabilization (MOMP), the mitochondria release a number of factors that are critically involved in cell death signaling (Tait and Green, 2010). Bcl-2 family members are regarded as the key regulators of mitochondria-dependent apoptosis (Moldoveanu et al., 2014); however, dynamin-related protein 1 (Drp1), which orchestrates mitochondrial fission, also participates in apoptotic regulation by stimulating Bax oligomerization and thereby enhances MOMP (Montessuit et al., 2010); accordingly, the inhibition of Drp1 blocks mitochondrial fission and inhibits apoptosis (Cassidy-Stone et al., 2008). During mitochondrial fission, Drp1 assembles from the cytosol onto the mitochondria at focal sites of division, forming spiral chains around membrane constriction sites. It has been well documented that the phosphorylation/dephosphorylation of Drp1 may act as a molecular switch to “turn on” or “turn off”mitochondrial fission (Chang and Blackstone, 2010). The phosphorylation of Drp1 at Ser 656 by cyclicAMP-dependent protein kinase (PKA) induces mitochondrial elongation, whereas the dephosphorylation of Ser 656 by calcineurin promotes mitochondrial fragmentation (Cribbs and Strack, 2007). Calcineurin is a calciumand calmodulindependent phosphatase. In apoptotic cells, the fragmentation of depolarized mitochondria depends on Ca-evoked, calcineurin-mediated dephosphorylation of Drp1 at its conserved serine 637 site (Cereghetti et al., 2008). The importance of calcineurin in mitochondrial fission is also supported by the findings that an inhibitor of calcineurin (Cereghetti et al., 2010) or the use of a miRNA targeting calcineurin (Wang et al., 2011) regulates mitochondrial fission by modulating Drp1 dephosphorylation and translocation. In this study, we explored an uncanonical, lysosomal chymotrypsinmediated activation mechanism of calcineurin that leads to Drp1-mediated mitochondrial fission in apoptotic cells. Wehavepreviously reported that chymotrypsin is not only a digestive enzyme secreted by the pancreas but also expressed widely in rat tissues (Zhao et al., 2010) and cached in lysosomes (Miao et al., 2008). However, the expression and subcellular localization of chymotrypsin in cells with human origin remain unclear. Using immunofluorescence, we found that chymotrypsin was colocalized with the lysosomal marker protein LAMP1 in human neuroblastoma SH-SY5Y cells (Fig. 1A). Upon LeuLeuOMe treatment, which induced lysosomal membrane permeabilization (LMP) directly, lysosomal chymotrypsin was relocated to the cytosol. The induction of LMP triggered apoptosis in SH-SY5Y cells, which was evidencedby the releaseofmitochondrial cytochrome c (Fig. 1B), the cleavage of PARP by activated caspase 3 (Fig. 1C), and the increase in the percentage of apoptotic cells with sub-G1 DNA content (Fig. 1D). The pretreatment of cells with TPCK, which is a specific chymotrypsin inhibitor, effectively inhibited caspase 3 activation and prevented apoptosis, suggesting that lysosomal chymotrypsin may be responsible for the LMPtriggered apoptosis. To further confirm that the translocation of chymotrypsin to the cytosol was sufficient to induce apoptosis, we introduced recombinant human chymotrypsin into the cytosol of SH-SY5Ycells with theBioPorter reagent and found that the intracellular delivery of chymotrypsin significantly potentiated apoptosis, suggesting that chymotrypsin plays a proapoptotic role (Fig. 1E). We found that mitochondrial fission was an event that occurs downstream of LMP in early stage apoptotic cells. Confocal microscopic images indicated that the mitochondria tended to fuse when the lysosomes are intact, whereas 6-h LeuLeuOMe treatment, which induced LMP, ultimately led to mitochondrial fission (Fig. 1F). Pretreatment with TPCK partially blocked the fission of mitochondria (Fig. 1G and 1H); however, E64d and pepstatin A, two inhibitors of the lysosomal cathepsins, had no apparent effect on the LeuLeuOMe-induced mitochondrial fission and apoptosis (data not shown). These data suggested the involvement of chymotrypsin in mitochondrial fission during apoptosis. Mitochondrial fission depends on the translocation of cytoplasmic Drp1 to mitochondria, where it binds to Fis1, oligomerizes, and constricts the organelle, ultimately leading