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SMAC/Diablo is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in-vivostudy, a survivin/BIRC5-derived peptide was selected due to its overexpression in many cancers, and involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD8+ cells and increased cell-intrinsic PD-1/PD-L1 expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents a innovative therapeutic cancer paradigm.

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5′-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase.

Abstract.: A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca2+ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl2, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca2+ at a submicromolar concentration, the isolated thiol-protease has lost the Ca2+ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca2+-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94.

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [a-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [a-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP ÃDP>GTP?S>ADP ßS, UTP, 2MeSATP, AMP-PNP >AMP-PCP>AMP>adenosine; for p96 it is: ADP?ADP ß S ? ATP » AMP-PCP, AMP-PNP>GTP?S? AMP>2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADPßS, UTP? 2MeSATP, GTP?S, AMP-PNP, ATP?ADP>AMPPCP>adenosine>AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.

Dicyclohexylcarbodiimide (DCCD), a hydrophobic carboxyl reagent, inhibited Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles, induced by elevated pH, tetraphenylboron, ATP + Pi, or membrane modification with acetic anhydride. Under the conditions used, the same concentrations of DCCD were required for inhibition of Ca2+ release, Ca2+-ATPase activity, and Ca2+ uptake. On the other hand, free Ca2+ or alkaline pH prevented the inhibition by DCCD of Ca2+-ATPase and coupled Ca2+ transport but not that of Ca2+ release. Moreover, several hydrophilic carboxyl reagents inhibited Ca2+-ATPase but not Ca2+ release. We suggest that a carboxyl residue(s), located in a hydrophobic region of a protein(s), is involved in the control of Ca2+ release, where DCCD interaction with this group blocks Ca2+ release. This group is distinct from the one involved in the inhibition of Ca2+-ATPase. DCCD also inhibited [3H]ryanodine binding to junctional sarcoplasmic reticulum membranes. The presence of Ca2+ or an alkaline pH only slightly affects the degree of inhibition of ryanodine binding by DCCD. Incubation of the membranes with [14C]DCCD resulted in labeling of 350-, 170-, 140-, 53-, and 30-kDa proteins in addition to the Ca2+-ATPase. The involvement of one or all of the DCCD-labeled proteins in Ca2+ release and ryanodine binding is discussed.

Abstract: The association of an endogenous, Ca2+-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and beta-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.1 mgrm and maximal activity at 10 mgrm. Mg2+ is also an activator of this enzyme (CI50=22 mgrm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type.This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca2+-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. A possible involvement of this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity is discussed.

Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2+ accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2+ accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca2+ accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5′-[beta gamma-methylene]triphosphate or adenosine 5′-[beta gamma-imido]triphosphate. The presence of Ca2+, but not Mg2+, during the preincubation, prevents the effect of ATP + Pi on Ca2+ accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2+ efflux from actively or passively loaded vesicles. This Ca2+ efflux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[alpha-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5′-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).

Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.