Rodney P. Guttmann

Modulation of the in Situ Activity of Tissue Transglutaminase by Calcium and GTP

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situtransglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r 2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly lessin situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTGin situ.

Distinct cleavage patterns of normal and pathologic forms of α‐synuclein by calpain I in vitro

Parkinsons disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha‐synuclein (α‐syn), the protein mutated in some patients with familial PD. The mechanisms of α‐syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave α‐syn in vitro. Calpain I cleaved wild‐type α‐syn predominantly after amino acid 57 and within the non‐amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized α‐syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild‐type α‐syn after amino acid 57, this did not occur in mutant A53T α‐syn. This paucity of proteolysis could increase the stability of A53T α‐syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild‐type α‐syn. However, once α‐syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of α‐syn by cleaving at aberrant sites within the C‐terminal region. Elucidating the role of calpain I in the proteolytic processing of α‐syn in normal and diseased brains may clarify mechanisms of neurodegenerative α‐synucleinopathies.

NMDA receptor pharmacology: perspectives from molecular biology.

The NMDA receptor is an important target for drug development, with agents from many different classes acting on this receptor. While the severe side effects associated with complete NMDA receptor blockade have limited clinical usefulness of most antagonists, the understanding of the multiple forms of NMDA receptors provides an opportunity for development of subtype specific agents with potentially fewer side effects. Different NMDA receptor subtypes are assembled from combinations of NR1 and NR2 subunits with each subunit conveying distinct properties. The NRI subunit is the glycine binding subunit and exists as 8 splice variants of a single gene. The glutamate binding subunit is the NR2 subunit, which is generated as the product of four distinct genes, and provides most of the structural basis for heterogeneity in NMDA receptors. Pharmacological heterogeneity results from differences in the structure of ligand binding regions, as well as structural differences between subtypes in a modulatory region called the LIVBP-like domain. This region in NR1 and NR2B controls the action of NR2B-selective drugs like ifenprodil, while this domain in receptors containing the NR2A subunit controls the action of NR2A-selective drugs such as zinc. This suggests that NMDA receptor subtype selective drugs can be created, and further understanding of subtype specific mechanisms ultimately may allow successful use of NMDA receptor antagonists as therapeutic agents.

Calpain-dependent Endoproteolytic Cleavage of PrPSc Modulates Scrapie Prion Propagation

Previous studies using post-mortem human brain extracts demonstrated that PrP in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27–30), the protease-resistant core of PrPSc (1). The role of this endoproteolytic cleavage of PrP in prion pathogenesis and the identity of the cellular protease responsible for production of the C2 cleavage product has not been explored. To address these issues we have taken a combination of pharmacological and genetic approaches using persistently infected scrapie mouse brain (SMB) cells. We confirm that production of C2 is the predominant cleavage event of PrPSc in the brains of scrapie-infected mice and that SMB cells faithfully recapitulate the diverse intracellular proteolytic processing events of PrPSc and PrPC observed in vivo. While increases in intracellular calcium (Ca2+) levels in prion-infected cell cultures stimulate the production of the PrPSc cleavage product, pharmacological inhibitors of calpains and overexpression of the endogenous calpain inhibitor, calpastatin, prevent the production of C2. In contrast, inhibitors of lysosomal proteases, caspases, and the proteasome have no effect on C2 production in SMB cells. Calpain inhibition also prevents the accumulation of PrPSc in SMB and persistently infected ScN2A cells, whereas bioassay of inhibitor-treated cell cultures demonstrates that calpain inhibition results in reduced prion titers compared with control-treated cultures assessed in parallel. Our observations suggest that calpain-mediated endoproteolytic cleavage of PrPSc may be an important event in prion propagation.

Specific proteolysis of the NR2 subunit at multiple sites by calpain

The NMDA subtype of glutamate receptor plays an important role in the molecular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calcium‐dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cotransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cellular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immunoblot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C‐terminal region. In vitro calpain digestion of fusion protein constructs containing the C‐terminal region of NR2A yielded two cleavage sites at amino acids 1279 and 1330. Although it has been suggested that calpain cleavage of the NMDA receptor may act as a negative feedback mechanism, the current findings demonstrated that calpain cleavage did not alter [125I]MK801 binding and that receptors truncated to the identified cleavage sites had peak intracellular calcium levels, 45Ca uptake rates and basal electrophysiological properties similar to wild type.

Oxidation Inhibits Substrate Proteolysis by Calpain I but Not Autolysis

In this study, the effects of oxidation on calpain I autolysis and calpain-mediated proteolysis were examined. Calpain I was incubated with increasing concentrations of free calcium in the presence or absence of oxidant, and autolytic conversion of both the 80- and 30-kDa subunits was measured by immunoblotting utilizing monoclonal antibodies which recognize both autolyzed and non-autolyzed forms of each subunit, respectively. Autolytic conversion of the 80-kDa subunit of calpain I was not detected until free calcium concentration was greater than 40 μM, whereas autolysis of the 30-kDa subunit did not occur until the free calcium concentration was greater than 100 μM. In addition, autolytic conversion of either the 80- or 30-kDa subunit was not inhibited by the presence of oxidant. Calpain I activity was measured using the fluorescent peptide N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin or the microtubule-associated protein tau as substrate. Calpain I was found to have proteolytic activity at free calcium concentrations below that required for autolysis. Calpain I activity was strongly inhibited by oxidant at all calcium concentrations studied, suggesting that proteolytic activity of both the non-autolyzed 80-kDa and autolyzed 76-kDa forms was susceptible to oxidation. Interestingly, whereas oxidation did not inhibit autolytic conversion, the presence of high substrate concentrations did result in a significant reduction of autolysis without altering calpain proteolytic activity. Calpain I activity that had been inhibited by the presence of oxidant was recovered immediately by addition of the reducing agent dithiothreitol.

Oxidative Stress Inhibits Calpain Activity in Situ

In this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrateN-succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridineN-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo.

Frataxin point mutations in two patients with Friedreich's ataxia and unusual clinical features

Two patients with a progressive ataxia are presented with clinical features consistent with classic Friedreichs ataxia (FRDA), but also with features unusual for FRDA. Analysis of DNA showed that each patient is heterozygous for the expanded GAA repeat of FRDA, but carries a base change on his other frataxin allele. For one patient a non-conservative arginine to cysteine amino acid change is predicted at amino acid 165 whereas the other mutation is found at the junction of exon one and intron one. Muscle biopsy showed an absence of frataxin immunoreactivity in the patient harbouring the intronic mutation, confirming the pathological nature of the base change. These mutations extend the range of point mutations seen in FRDA, and agree with recent reports suggesting phenotypic variation in patients with FRDA harbouring point mutations in conjunction with an expanded GAA repeat.

Tissue Transglutaminase Is an In Situ Substrate of Calpain: Regulation of Activity

Abstract: Tissue transglutaminase (tTG) is a calcium‐dependent enzyme that catalyzes the transamidation of specific polypeptide‐bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium‐activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine‐5′‐O‐(3‐thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH‐SY5Y cells with 1 nM maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an ∼50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.

Identification and characterization of PEBP as a calpain substrate

Calpains are calcium‐ and thiol‐dependent proteases whose dysregulation has been implicated in a number of diseases and conditions such as cardiovascular dysfunction, ischemic stroke, and Alzheimers disease (AD). While the effects of calpain activity are evident, the precise mechanism(s) by which dysregulated calpain activity results in cellular degeneration are less clear. In order to determine the impact of calpain activity, there is a need to identify the range of specific calpain substrates. Using an in vitro proteomics approach we confirmed that phosphatidylethanolamine‐binding protein (PEBP) as a novel in vitro and in situ calpain substrate. We also observed PEBP proteolysis in a model of brain injury in which calpain is clearly activated. In addition, with evidence of calpain dysregulation in AD, we quantitated protein levels of PEBP in postmortem brain samples from the hippocampus of AD and age‐matched controls and found that PEBP levels were approximately 20% greater in AD. Finally, with previous evidence that PEBP may act as a serine protease inhibitor, we tested PEBP as an inhibitor of the proteasome and found that PEBP inhibited the chymostrypsin‐like activity of the proteasome by ∼30%. Together these data identify PEBP as a potential in vivo calpain substrate and indicate that increased PEBP levels may contribute to impaired proteasome function.