Dissertations / Theses on the topic 'Calpain system'

Thesis (M.S.)--West Virginia University, 1999.
Title from document title page. Document formatted into pages; contains vii, 93 p. : ill. (some col.) Includes abstract. Includes bibliographical references (p. 72-80).

In an attempt to further our understanding of the relationship between the calpain system and postmortem tenderization, three muscles [biceps femoris (BF), infraspinatus (IS), and longissimus (LD)] from normal (N = 6) and callipyge (N = 6) sheep were studied. Callipyge is a genetic phenomenon where carriers of the callipyge gene present a hypertrophy of pelvic and torso muscles, such that BF and LD are affected while IS is not. It has been observed characteristically that calpastatin and m-calpain activities are increased in muscles of animals affected by the callipyge phenotype. Soluble calpain and calpastatin, and myofibril-bound μ-calpain activities, and myofibrillar fragmentation index (MFI) were determined at death, 1d, 3d and 10d postmortem. Sarcomere length was determined at 1d and 10d postmortem. Shear force of the longissimus muscle was determined at 1d, 3d and 10d postmortem. Western blots for calpastatin, μ-calpain, desmin, nebulin, titin, troponin-T and α-actinin were performed to follow the degradation pattern of those proteins. The calpastatin and m-calpain activities were more than two-fold greater in BF and LD muscles from callipyge than in the same muscles from normal animals. Calpastatin activities in infraspinatus muscle from normal animals were higher than in the other two muscles of this phenotype. Soluble μ-calpain activities were higher at death for normal phenotype in BF and IS muscles and it decreased rapidly during postmortem storage. However, the rate of this decrease in that activity was faster in normal than in callipyge phenotype. Myofibrils contained calcium dependent protease activity and this activity was inhibited by cysteine proteases inhibitors and by calpastatin to some degree. There was no difference in the myofibril-calcium dependent protease activity between phenotypes at any time postmortem, presenting lower activity at death. The magnitude of protein degradation and tenderization were assessed by MFI and shear force, respectively. Neither the MFI nor shear force changed appreciably during storage of the callipyge affected muscles. Calpastatin level seems to determine the rate of postmortem tenderization.

2010/2011
The calpains are a family of intracellular cysteine proteases, among which the best studied isoforms, micro- (CAPN1) and milli-calpain (CAPN2), are heterodimers consisting of a catalytic subunit and a common regulatory subunit, CAPNS1, required for function. Calpain is involved in many processes important for cancer biology, such as autophagy, indeed in calpain-depleted cells autophagy is impaired, with a subsequent increase in apoptosis sensitivity. Calpain is also important in all the stages of the stress response. A proteomic approach was employed for the identification of novel CAPNS1 interacting proteins. Proteins immunoprecipitating with endogenous CAPNS1 in HT1080 cell lysates were analyzed by Mass Spectrometry. We identified novel partners among which the deubiquitinating enzyme USP1, a key regulator of the DNA damage response and genome integrity maintenance via its specific action on FANCD2, involved in DNA repair and protection from chromosome instability, and PCNA, involved in the regulation of translesion DNA synthesis (TLS), that bypasses DNA lesions with low stringency basepairing requirements. We performed co-IP assays in lysates of 293T cells and confirmed that the interaction was specific. Furhermore, we observed that calpain is able to bind a USP1 C-terminal deleted mutant, suggesting that USP1 first 523 aminoacids were sufficient for the binding. To understand what is the effect exerted by calpain upon USP1, we depleted calpain activity in a series of cell lines, and followed the fate of endogenous USP1. We transfected CAPNS1 specific siRNAs, or treated cells with a specific inhibitor of calpain, and we observed a strong decrease in USP1 protein levels. This effect should be at a post-transcriptional level, since any significant change in USP1 mRNA levels is detected. We also obtained the same result by transfecting a siRNA specific for CAPN1, the gene encoding for the catalytic subunit micro-calpain. Moreover, we studied the role of calpain in the PCNA-mediated switch between high fidelity replication and TLS upon UV irradiation. In mouse embryonic fibroblasts knockout for CAPNS1, USP1 downregulation is coupled to an increase in PCNA monoubiquitination. Moreover, CAPNS1-depleted U2OS cells showed an increase in the percentage of nuclei containing PCNA-induced foci upon UV irradiation. Since we demonstrated that calpain can modulate an important regulator of DNA damage response such as USP1, we investigated if calpain could have a role in genome integrity maintenance. CAPNS1 depleted cells showed a reduced rescue in DNA repair compared to control cells, suggesting that increased levels in PCNA monoubiquitination could lead to an increased amount of errore-prone TLS. Calpain plays an important role in autophagy, so we asked if USP1 degradation in absence of calpain activity could involve autophagic pathways. We first blocked macroautophagy by silencing ATG5, and we observed that USP1 was downregulated, suggesting that the depletion of ATG5 could lead to an increased activity of other degradation pathways. To impaire chaperone-mediated autophagy (CMA), we silenced a protein important for autophagosome formation, LAMP-2A. Also in this case we observed a decrease in USP1 protein levels, thus suggesting that USP1 is alternatively degraded by different pathways. However, we observed that USP1 is stabilized upon inhibition of lysosomal enzymes, suggesting that USP1 may be degraded in the lysosome. To better understand the mechanism by which calpain affect USP1 stability we search for an effect of calpain upon USP1 co-factor and activator UAF1/WDR48. CAPNS1-depleted cells showed WDR48 downregulation, but WDR48 overexpression only partially rescue USP1 protein levels in this cells. Furthermore, we provided evidences that calpain regulation of p35/p25 activator of Cdk5 can affect Cdh1 phosphorylation and thus APC/Cdh1 activity, leading to a regulation of USP1 stabilization. In conclusion, we identified USP1 as a novel interactor of calpain, and we found that calpain is important for USP1 stability, since in its absence USP1 is downregulated. The importance of this novel regulation is strengthened by the recent findings that unveiled a role of USP1 in maintenance of a mesenchymal stem cell program in osteosarcoma, and thus placing calpain in a crucial regulatory position for cancer development.
XXIV Ciclo
1983

Calpains, a highly conserved family of calcium-dependent cysteine proteases, are divided in two groups; classical and non-conventional calpains. Calpain-1 and calpain-2, the classical ones, are ubiquitously expressed and abundant in the CNS. Findings through different experimental approaches, predominantly pharmacological calpain inhibitors, proposed the necessity of the proteases for the modulation of various biological events particularly in the CNS, or a functional link between calpain and neurodegeneration. Significant functions associated with calpain activity are neuronal proliferation/differentiation, signal transduction, apoptosis, and synaptic plasticity; or neuronal death in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ischemic stroke. However, due to limited insights of the approaches taken, such as non-specificity of the inhibitors, the exact roles of calpains in the CNS and the key mechanisms underlying them remain controversial. Calpain-1/calpain-2 germline knock-out are embryonic lethal at a very early stage hindering the use of these lines as mouse models for CNS studies. Accordingly, this thesis research introduced a unique brain-specific calpain-1/calpain-2 knock-out and explored the role of the proteases in brain development/function and in neuronal death. The first set of analyses examined how the elimination of calpain-1/calpain-2 activities in mouse brain impacts CNS development in general and synaptic plasticity in CA1 neurons of hippocampus. CNS-specific elimination of CAPNS1, the common small subunit, abolished calpain-1/calpain-2 activities in mouse brain. In contrast to Calpain-1/calpain-2 germ line knock-outs, the brain-specific knock-outs are viable and the general development of mouse brain is normal. However, morphology of dendrites in pyramidal neurons of the hippocampal CA1 region showed significantly decreased dendritic branching complexity and spine density. Consistent with dendrite morphological abnormalities, electrophysiological analyses revealed a significant decrease in field excitatory postsynaptic potentials, long term potentiation, and learning and memory in the hippocampal CA1 neurons of the mutants. In the second part of this research we investigated the direct role of the calpains in neuronal death and their potential downstream targets in in vitro models of PD and ischemic stroke. Our findings indicated that ablation of calpains activity improves survival of different types of neurons against mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), glutamate, and hypoxia. Importantly, we demonstrated an increase in p35-cleavage to p25, a cyclin dependent kinase 5 (Cdk5) activator, and that restoration of p25 significantly suppresses the neuronal survival associated with calpain deficiency. Taken together, this work unequivocally establishes two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

The objective of this study was to evaluate the impact of an anabolic implant and its use with the beta-adrenergic agonist zilpaterol hydrochloride (ZH) the calpain proteolytic system activity across specific areas of the beef strip steak over two aging days. Crossbred heifers were blocked by weight and randomly assigned to one of three treatments: 1) no implant or ZH (CON), 2) implant, no ZH (IMP), and 3) implant and ZH (IMP+ZH). At slaughter, strip steaks were collected and aged for 3 and 14 d. Samples were evaluated for Minolta objective color scores, pH, bioelectrical impedance analysis, and were then cut into lateral, lateral/medial, and medial sections. Protein was extracted from each section, and the calpain proteolytic system was evaluated. A day of aging effect was seen in protein degradation, along with a treatment by section interaction proteolytic activity. No differences were found in pH or color by treatment.

The absolute clarity of the lens of the eye is vital in the visual system. The unique structural and physiological properties of the lens are tightly integrated with highly ordered protein content to allow the lens to remain transparent. Consequently, any alteration or disturbance of these highly ordered proteins can affect the optical properties of the lens. In humans, cataracts are the major cause of blindness, yet the exact aetiology of cataract formation (cataractogenesis) is not fully understood. The purpose of the current research was to investigate whether deregulation of the Ca²⁺-dependent enzyme, calpains, following changes in lens Ca²⁺ homeostasis, is a key mechanism leading to undesired cleavage of a number of proteins that are linked with maintaining lens transparency and contributing to cataractogenesis. An ovine lens culture (in vitro) system and the heritable ovine cataract (in vivo) model were used to test the research hypothesis. The Ca²⁺ ionophore, ionomycin, was used to induce a Ca²⁺ overload and in vitro opacification during lens culture. Opacity in the lens was graded by a computer image analysis program. Protein profile (SDS-PAGE, 2-DE and Western detection), calpain activity (casein zymography), lens structure (microscopic view) and cytotoxicity level (LDH leakage assay) were analysed in Ca²⁺-induced opaque lenses. The involvement of calpain during opacification was further examined by applying synthetic exogenous calpain inhibitors to the in vitro system. Two novel exogenous calpain inhibitors were also assessed for their therapeutic potential in preventing the progression of cataracts in the in vivo cataract model by topical administration of the inhibitor direct to the sheep's eye over a 11 week period. HPLC was used to detect the penetration of these compounds into ocular tissues. Sustained Ca²⁺ influx into cultured lenses caused dense opacification. The opacity was characterised by formation of a turbid fraction and cell death in the outer cortex of the ovine lens. There was increased calpain autolysis associated with the progress of opacification, indicating increased calpain activity. Major degradation of the cytoskeletal proteins, spectrin and vimentin, was observed whilst there was limited degradation of the lens structural soluble proteins, crystallins, in response to a Ca²⁺ flux. Lens proteins were protected from degradation by adding synthetic calpain inhibitors to the culture medium. Topical administration of novel anti-calpain molecules failed to retard the progression of cataractogenesis in the ovine inherited cataract model. Further investigation of drug penetration showed that efficacy of inhibitory compounds was limited by permeability of these molecules across the cornea and the ability of the molecules to reach and penetrate into the lens. The ovine lens Ca²⁺-induced opacification (OLCO) model in this thesis has provided a model to understand the role of Ca²⁺ homeostasis in lens transparency. With sustained intracellular Ca²⁺ level, the degradation of cytoskeletal elements is highly correlated with calpain activity. Cataractogenesis is the pathological response to the loss of lens Ca²⁺ homeostasis in this model. The current results support the hypothesis that the deregulation of calpain activity is a trigger for a series of cascading events, leading to death of the cells in the lens.

Muscle atrophy is a serious side effect seen with extended time in space. Proteolytic degradation of specific muscle proteins leads to smaller, weaker muscles that are structurally more susceptible to damage. Calpains are proteases that specifically degrade target proteins of the myofibril, and have been implicated in many types of muscle atrophy. Calpain activity is regulated by a combination of activation by calcium and inhibition by calpastatin, its endogenous inhibitor. This dissertation describes the generation of a skeletal muscle-specific, doxycycline (Dox) controlled, calpastatin over-expression system in transgenic mice to regulate calpain activity. A dual construct system, the transactivator line utilizes an optimized tet-on system and a modified muscle creatine kinase promoter to create muscle specific expression of a tet transactivator. The second transgenic line, consisting of a bi-directional promoter centered on a tet responder element controlling both a luciferase reporter gene and a tagged calpastatin, is transcriptionally silent until activated by a dox induced transactivator molecule. Compound hemizygous mice showed high level, Dox dependent, muscle-specific overexpression of luciferase and transgenic calpastatin, demonstrating the effectiveness and flexibility of the tet-on system to provide a tightly regulated overexpression system in adult skeletal muscle. Consistent overexpression of calpastatin was hard to maintain, however, and not all of the proposed experiments could be achieved. Additional studies compared the degradation of hindlimb suspended mouse muscle proteins (weightlessness model) to those of ground control muscle proteins briefly incubated in Ca²⁺ (to initiate calpain degradation). Four proteins known as targets for calpain degradation were selected for analysis. Degradation responses of myofibrillar proteins titin, nebulin, and troponin T in hindlimb suspension clearly mimicked that seen with the calcium incubations. The cytoskeletal calpain target protein, desmin, however did not respond the same to both treatments showing moderate degradation with calcium and no degradation with hindlimb suspension. These data suggest that myofibrillar calpain target proteins, but not necessarily cytoskeletal proteins, are rapidly targeted for degradation in hindlimb suspension in a manner similar to that induced by calcium, implicating calpain as a mediator of this degradation.

Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

碩士
東海大學
畜產學系
87
The growth rate of skeletal muscle dependents on the rates of muscle protein synthesis and protein degradation. It was reported that calpains played an important role on the initiation of muscle protein degradation. The calpains depend on its calcium requirement can be grouped into - and m-calpain. Both and m-calpain contain two subunit, small subunit ( CS; 30KDa ) and large subunit ( CL; 80KDa ). The objectives of this study were to investigate the function of CS by expressing the autolysis form of CS in L8 myoblast. Rat post-autolysis small subunit ( 21KCS; 21KDa ) cDNA was amplified by polymerase chain reaction ( PCR ). The cDNA ( 577bp ) then was inserted into pMAMneo-SV40 ( pSV ) vector. The new plasmid is referred to pMAMneo-SV40-Small subunit ( pSV-SS ). The plasmid pSV and pSV-SS were transfected into L8 myoblasts, respectively. Then the single cloning was selected by G418 ( 300g/ ml ) containing medium. After selection, the transfected cell were analyzed by PCR using Neor primer against the genomic DNA and stable cloning cell line L8-Neo, SS1, SS2 and SS3 were established. CS mRNA were analyzed by Reverse transcription polymerase chain reaction ( RT-PCR ) in transfected and control cells using primer 21K-1 and oligo-dT. The data indicated that SS2 and SS3 had expression of exogenous CS mRNA ( 21KCS mRNA ), however, SS1 did not express the exogenous CS mRNA. In addition, and m-calpain were analyzed by Western blot in total and membrane protein. In analysis of total protein, the results showed that the protein concentration of - calpain in SS2 and SS3 were 15.7% and 17.3% higher than control ( L8-Neo ), respectively. The protein concentration of m-calpain in SS2 and SS3 were 23.3% and 16.6% higher than control ( L8-Neo ), respectively. In addition, SS3 and control ( L8-Neo ) were analyzed by immunoprecipitation to detect the stability of calpain. The data indicated that the stability of -calpain in SS3 was 36.3 hours higher than control ( 31.9hours ). The stability of -calpain was 40.5 hours higher than control ( 36.3hours ). In analysis of membrane calpain concentration, the transfected cells ( SS1、SS2 and SS3 ) and control cell ( L8-Neo ) have no significant difference by expression of exogenous CS. Also, there is no different in total protein degradation between transfected cells and control cells. These results imply that expression of exogenous 21KCS can increase the protein level of - and m-calpain and increase the stability of calpains. The result indicated that 21KCS effect on translation level but not on transcription level. On the pther hand, 21KCS can not improve the opportunity to translocte to the membrane or change the rate of protein degradation in myoblast of rat.

The purpose of this study was to exainine the relationship between the activation of the calcium stimulated cysteine protease, calpain, its endogenous inhibitor, calpastatin, and myofibrillar protein composition (troponin I (Tnl), troponin T (TnT) and tropomyosin (TM)) in an exercise-induced muscle damage model. It was hypothesized that this protease system initiated skeletal myofibrillar protein loss (and perhaps subsequent peptide release) from the contractile apparatus. In addition, lowering calpain activity (by the use of an exogenous inhibitor) was hypothesized to attenuate the composition of myofibrillar protein substrates for calpain (i.e. Tnl, TnT, TM). To test these hypotheses, male Wistar rats (~315g) were randomly assigned to one of six groups: 1) control sedentary (n=8), 2) control + cysteine protease inhibitor (E64c)(n=8), 3) running (25 meters/minute (-16°) for 45 min.)(n=8), 4) running + E64c (n=8), 5) running + 6 nr. recovery (n=8), or 6) running + 6 nr. recovery + E64c (n=8). Calpain I and II isoforms were isolated from rat hind-limb skeletal muscles, purified via phenyl-sepharose chromatography and their activities quantified using a casein-release assay. Calpastatin was isolated by a heat-release procedure and assayed based on its ability to inhibit calpain. Finally, myofibrillar proteins were resolved from a muscle homogenate via SDS-PAGE and their concentrations quantified using computer densitometry. Calpain I and II activities increased by 36.1% and 37.5% respectively, immediately following exercise, and at 6 hours of recovery were 16.4% and 15.9% compared to controls (p<0.05). With E64c administration, the run-induced activation of calpain I (3.7%) and II (8.2%) following exercise was much less, which was maintained into the recovery (5.3% and 9.7% respectively)(p>0.05). Calpastatin activity did not change with exercise, however at the 6 hour recovery, activity was elevated by 74.8% (p<0.05). With E64c injection, this activity increase remained elevated over control levels (97.4%)(p<0.05). The proportion of bands corresponding to myofibrillar sTnl, sTnT and T M decreased to 75.1%, 75.6% and 90.1% of control values respectively, immediately following exercise (p>0.05) and at 6 hours of recovery were 73.2%, 75.4% and 80.3% (p>0.05). With E64c injection, any exercise-induced loss of sTnl (108.0% of control), sTnT (105.5% of control) or TM (110.7% of control) following running was arrested (p>0.05). This trend was maintained into the recovery (104.2%, 101.8% and 113.8% respectively)(p>0.05). The proportion of cytosolic protein bands corresponding to sTnl, sTnT and TM increased to 363.8%, 343.5% and 430.1% of control respectively, immediately after exercise (p<0.05) and at 6 hours of recovery were 386.4%, 372.7% and 473.2% (p<0.05). With E64c administration, the exercise-induced increase in sTnl (532.3% of control), sTnT (509.0% of control) and TM (478.6% of control) was enhanced (p<0.05). This change remained into the recovery (537.4%, 530.7% and 514.9% respectively)(p<0.05). It is concluded, based on E64c's ability to attenuate both the exercise-induced activation of calpain and myofibrillar protein loss/breakdown, that calpain is a causative factor for protein composition changes in vivo. Calpastatin also dominates the protease system during recovery, shifting the environment to one of protein maintenance.

The first goal of this study was to understand the role of u-calpain in skeletal muscle protein degradation in cultured muscle cells. Several strategies were developed to down-regulate endogenous u-calpain activity and m-calpain activity in rat myotubes. These included over-expression of antisense u-calpain (AnsL), dominant negative u-calpain (DN-u-CL), antisense 30K subunit (AnsS) and fused antisense u-calpain/30K (AnsLS, i.e., 80K/30K). The ability to regulate calpain activity was confirmed by fodrin degradation (an index of calpain activity). Our data supported the contention that u-calpain contributes significantly to total protein degradation in myotubes. Specifically, over-expressing DN-u-calpain reduced total protein degradation by 7.9% (P<0.01) at 24 hr time point and by 10.6% (P<0.01) at a 48 hr time point. Similarly, over-expression of antisense u-CL and the 30K subunit reduced total protein degradation significantly at the 24 hr time point (P<0.05). However, over-expression of the fused antisense (80K/30K) did not affect (P>0.05) the total protein degradation. In addition to this we determined that desmin was a calpain substrate and that calpain could not degrade tropomyosin. The second goal of this study was to evaluate the relationships among u- and m-calpain and the 30KD subunit. The rationale for this study was that our earlier work indicated coordinated regulation of the calpain subunits. Our data demonstrated for the first time that the transcription and translation of u-calpain and 30K, and m-calpain and 30K are coordinately regulated, respectively. However, the expression of u-calpain did not affect the expression of m-calpain The third goal of this study was to develop a skeletal muscle-specific inducible expression system that may be used in transgenic animal research. A skeletal muscle a-actin promoter was used to replace the cytomegalovirus immediate-early promoter (pCMV) in the ecdysone inducible mammalian expression system. LacZ was used as a reporter gene. A beta-galactosidase staining assay and high-sensitivity B-gal activity assay indicated that the skeletal muscle-specific expression system functioned in myotubes. After 48 hr of administration of ponasterone A (inducer), the treated cells had 15-fold higher B-gal activity than the control cells.
Graduation date: 2002

Tese de doutoramento em Biologia Experimental e Biomedicina, na especialidade de Neurociências e Doença, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra
Ischemic stroke is characterized by a decrease in oxygen supply to the brain with a consequent impairment in the metabolic activity. The sudden decrease in the levels of ATP affect the balance between excitatory and inhibitory neurotransmission, and the overactivation of glutamate receptors leads to neuronal death (excitotoxicity). Under these conditions, the [Ca2+]i overload to the postsynaptic cell induces the activation of calpains, a group calcium-dependent proteases that regulate the function of many substrate proteins through limited proteolysis. In parallel to calpain activation, the Ubiquitin-Proteasome System (UPS), one of the major protein degradation systems in the cells, which also regulate several key and physiological functions, was also shown to be downregulated under energy-depriving conditions. However, how these two proteolytic systems are interconnected under ischemic conditions is currently unknown. Multiple lines of evidences suggest that proteasome inhibition is beneficial in in vivo models of transient/global ischemia by halting the inflammatory process. However, proteasome activation may also be a powerful tool to prevent cell demise in acute brain injury, and in other disorders of the nervous system, by enhancing the degradation of damaged proteins. In this work, we characterized the alterations in the subcellular distribution of the proteasome in cultured neurons subjected to ischemic conditions, and investigated the mechanisms contributing to the downregulation of the proteasome under the same conditions. In particular, we focused on the putative effects of calpains on the proteasome components, and the impact of the proteasome in neuronal demise. We found that Oxygen-Glucose Deprivation (OGD), a well-known in vitro model mimicking transient global ischemia in cultured neurons, had a differential effect on the dendritic distribution of the proteasome proteins PSMA2 and Rpt6, used as markers of the 20S and 19S proteasome particles, respectively. Non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with antibodies against the PSMA2 and Rpt6 also showed a disassembly of the proteasome in cortical neurons subjected to in vitro ischemia, further indicating that this is an appropriate model to study the regulation of the proteasome under ischemic conditions. OGD was also found to enhance calpain activity (assessed by the cleavage of its substrate spectrin) and had the opposite effect on the activity of the proteasome (determined with a fluorogenic substrate). In contrast with the biochemical changes often associated with inhibition of the proteasome, polyubiquitin conjugates were found to be reduced in cortical neurons subjected to OGD, while no changes were detected in free ubiquitin levels. Two non-related assays were used aiming at identifying which subunits of the 19S proteasome may be cleaved by calpains: (i) cleavage of endogenous proteins in cortical neurons subjected to OGD and (ii) cleavage of green fluorescent protein (GFP) fusion proteins with proteasome subunits in extracts obtained from HEK293t cells incubated with recombinant calpain. We found that the ubiquitin acceptor Rpn10, together with Rpt3 and Rpt5 are calpain substrates, and this may also be the case for Rpt1 and Rpn3. Finally, here we report that inhibition of USP14, a deubiquitinating enzyme (DUB) associated with the 26S proteasome, provides robust neuroprotection to cerebrocortical neurons subjected to OGD. USP14 inhibition was previously shown to increase the proteolytic activity of the proteasome towards specific substrates, suggesting that increasing degradation of canonical proteasome substrates is sufficient to prevent cell death. Importantly, incubation of cortical neurons with IU1, the USP14 inhibitor, prevented the OGD-induced activation of calpains, but the underlying mechanism remains to be investigated. Taken together, the results show that OGD induces hypofunction of the proteasome, alongside with an increased activity of calpain against proteasome resident subunits. The mechanism proposed here conciliates the calpain-mediated cleavage of proteasome subunits with the observed disassembly under ischemic conditions. Enhancing the activity of the proteasome prevents cell demise associated with OGD in cultured cerebrocortical neurons, and may represent a novel therapeutic target to restore the deficits observed after stroke.
O acidente vascular cerebral (AVC) é caracterizado pela diminuição da irrigação sanguínea ao nível do cérebro, com consequente alteração da atividade metabólica. O súbito decréscimo dos níveis de ATP afeta toda a neurotransmissão excitatória e inibitória, e a sobreactivação dos recetores para o glutamato levam à morte neuronal (excitotoxicidade). Nestas condições, o aumento excessivo da [Ca2+]i na célula pós-sináptica é responsável por ativar as calpaínas, um grupo de proteases reguladas por este ião que controla a função de inúmeras proteínas na célula através de proteólise limitada. Paralelamente à ativação das calpaínas, o Sistema Ubiquitina-Proteassoma (UPS), o principal sistema de degradação proteico existente nas células, que tem um papel chave na regulação de vários processos biológicos, encontra-se inibido em condições de deficit de energia na célula. No entanto, a forma como estes dois sistemas proteolíticos interagem não é de todo conhecido. Várias evidências sugerem que a inibição do proteassoma tem um papel protetor em modelos in vivo de isquémia transiente/global, através da inibição do processo inflamatório. No entanto, a ativação do proteassoma também pode ser benéfica na prevenção da morte celular em várias agressões do tipo agudo/traumáticas ao nível do cérebro, bem como em doenças neurodegenerativas, através do aumento da degradação de proteínas danificadas. Neste trabalho, caracterizámos a alteração na distribuição subcelular do proteassoma em neurónios submetidos a isquémia in vitro, bem como os mecanismos que contribuem para a diminuição da atividade do proteassoma nas mesmas condições. Focámo-nos em particular nos efeitos putativos das calpaínas sobre componentes do proteassoma, bem como no impacto do UPS na morte celular. Observou-se que a deprivação de oxigénio e glucose (OGD) em neurónios em cultura, um modelo in vitro que mimetiza o efeito transitório da isquémia global, tem um efeito diferencial na distribuição dendrítica das proteínas PSMA2 e Rpt6, duas subunidades do proteassoma vulgarmente usadas como marcadores do 20S e do 19S, respetivamente. Eletroforese em géis de poliacrilamida em condições não desnaturantes, seguido de imunoblot contra as proteínas PSMA2 e Rpt6, revelou que o proteassoma é desmontado em neurónios corticais submetidos ao modelo in vitro de isquémia cerebral, indicando que se trata de um modelo apropriado para estudar a regulação do proteassoma nestas condições. A incubação transitória de neurónios em cultura na ausência de oxigénio e glucose induziu a ativação de calpaínas (avaliado através da análise da clivagem da espectrina) e alterou de forma oposta a atividade do proteassoma (determinado através de um ensaio fluorogénico). No entanto, ao contrário das alterações bioquímicas observadas após inibição química do proteassoma, os níveis de proteínas conjugadas com cadeias de poliubiquitina diminuiu em neurónios corticais em cultura submetidos a OGD, não tendo sido observadas quaisquer alterações nos níveis da ubiquitina livre. Com o objetivo de identificar que proteínas associadas ao 19S podem ser clivadas pelas calpainas, usámos dois ensaios distintos: (i) análise da clivagem de proteínas endógenas em neurónios corticais em cultura submetidos a OGD e (ii) clivagem de proteínas de fusão contendo a sequência de aminoácidos de subunidades do proteassoma associada a GFP (green fluorescent protein) em extratos de células HEK293t incubados com calpaína recombinante. Nestes ensaios observou-se a clivagem do recetor para a ubiquitina Rpn10, juntamente com a proteína Rpt3, sendo que o mesmo poderá também acontecer para as proteínas Rpt1 e Rpn3. Neste trabalho observámos também que a inibição da USP14, uma enzima de desubiquitinação (DUB) associada ao proteassoma 26S, tem um efeito protetor significativo em neurónios corticais submetidos a OGD. Estudos prévios mostraram que a inibição da USP14 estimula a capacidade proteolítica do proteassoma sobre substratos específicos, sugerindo que o aumento da degradação dos seus substratos canónicos é suficiente para prevenir a morte neuronal. Observou-se também que a incubação de neurónios corticais com o inibidor USP14 impede a ativação de calpaínas em resultado da incubação em condições de OGD. No entanto, os mecanismos responsáveis pela inibição das calpaínas nestas condições não estão esclarecidos. Em conjunto, os resultados obtidos mostram que a atividade do proteassoma se encontra diminuído em condições de OGD, bem como a clivagem de proteínas associadas ao mesmo por parte das calpaínas. O mecanismo aqui proposto concilia a clivagem de proteínas associadas ao proteassoma com a sua desmontagem em condições de isquémia. O aumento da atividade do proteassoma preveniu a morte de neurónios corticais em cultura submetidos a OGD, e pode representar uma nova estratégia terapêutica na recuperação do tecido nervoso após um AVC.