Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.     

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.     

Digestive versus regulatory proteases: on calpain action in vivo

Calpains, the cytoplasmic Ca2+-activated regulatory proteases, have no simple and clearly definable cleavage site specificity, which is in sharp contrast to digestive (e.g., pancreatic) proteases. For calpains, an approximate 10-aa segment having a variety of sequences and spanning the scissile bond, governs proteolytic cleavage. This permissivity is a precondition for calpains to act on several different substrate proteins in the cell. The specificity of calpain action may be ensured by anchoring/targeting proteins. Intriguingly, the established endogenous inhibitor protein, calpastatin, might also serve as a storage site. Furthermore, specificity may be encoded in the 'goodness' of the undecapeptide sequence in substrate proteins. Novel approaches are needed to reveal how calpains find their substrates in cells at the proper time and location.     

Heterogeneous nuclear ribonucleoprotein K interacts with and is proteolyzed by calpain in vivo

Calpain is a cytosolic "modulator protease" that modulates cellular functions in response to Ca2+. To identify in vivo substrates of calpain, yeast two-hybrid screening was done using the 5-EF-hand (penta-EF-hand; PEF) domain of the micro-calpain large subunit (domain IV), since several possible in vivo substrates for calpain have been previously reported to bind to the 5-EF-hand domains. Other than the regulatory subunit of calpain, which binds to the domain IV, heterogeneous nuclear ribonucleoproteins (hnRNP) K and R were identified, and shown to be proteolyzed by micro-calpain in vitro. When expressed in COS7 cells, hnRNP K and micro-calpain co-localized in the cytosol, and Ca2+-ionophore stimulation of the cells resulted in proteolysis of hnRNP K, indicating that hnRNP K is an in vivo substrate for calpain. Now, hnRNP K is considered to function as a scaffold protein for its binding proteins, such as PKCdelta and C/EBPbeta, which were reported to be calpain substrates, suggesting that hnRNP-K is a scaffold for calpain to proteolyze these proteins.     

Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo

Neuronal activity regulates the catabolism of specific structural proteins in adult mammalian brain. Pharmacological stimulation of rat hippocampal neurons by systemic or intraventricular administration of the excitatory amino acids kainate or N-methyl-D-aspartate induces selective loss of brain spectrin and the microtubule-associated protein MAP2, as determined by quantitative immunoblotting, but not of actin, the high molecular weight neurofilament polypeptide, or glial fibrillary acidic protein. The spectrin decrease occurs primarily by enhanced proteolysis, as levels of the major breakdown products of the alpha-subunit increase more than 7-fold. This proteolysis may occur from activation of the calcium-dependent neutral protease calpain I. The immunopeptide maps produced by alpha-spectrin degradation, selective loss of spectrin and MAP2, and decrease in calpain I levels are all consistent with calpain I activation accompanied by autoproteolysis. We propose that calcium influx and calpain I activation provide a mechanism by which neuronal activity regulates the degradation of specific neuronal structural proteins and may thereby modify neuronal morphology.     

In vivo administration of calpeptin attenuates calpain activation and cardiomyocyte loss in pressure-overloaded feline myocardium

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.     

Possible role of calpain I and calpain II in differentiating muscle

The variable distribution of the 80-kD subunit of two calcium-activated proteases, calpain I and calpain II, has been examined in L8 and L6 myoblasts, and their non-fusing variants, fu-1 and M3A using non-cross-reacting monoclonal antibodies to both subunits. Immunofluorescence results have shown that while the 80-kD subunit of calpain I is localized in the cytoplasm of all the myoblasts, the 80-kD subunit of calpain II appears to be predominantly associated with the plasma membranes of L8 and L6 myoblasts. The distribution of the 80-kD subunit of calpain II in non-fusing myoblasts, fu-1 and M3A, is generally cytoplasmic and diffuse. Immunoblot analysis of membrane and cytosol fractions of all the myoblasts using the monoclonal antibodies described above essentially confirms the immunofluorescence findings. Because calpain II exhibits a peripheral distribution in cells which are fusion-competent, L6 and L8 myoblasts, but not in fu-1 and M3A myoblasts, we suggest that calpain II may play a role in the Ca2+-mediated fusion events of differentiating (prefusion) myoblasts.     

A Model Implicating the Langerhans Cell in Keratinocyte Proliferation Control

A tentative model is presented which is based on existing data and our own cell kinetic and morphological observations in mice. The model suggests that the epidermal Langerhans cell plays a role in proliferation control of keratinocytes and may also act as an epidermal stem cell.     

Rosette nanotubes show low acute pulmonary toxicity in vivo

Nanotubes are being developed for a large variety of applications ranging from electronics to drug delivery. Common carbon nanotubes such as single-walled and multi-walled carbon nanotubes have been studied in the greatest detail but require solubilization and removal of catalytic contaminants such as metals prior to being introduced to biological systems for medical application. The present in vivo study characterizes the degree and nature of inflammation caused by a novel class of self-assembling rosette nanotubes, which are biologically inspired, naturally water-soluble and free of metal content upon synthesis. Upon pulmonary administration of this material we examined responses at 24 h and 7d post-exposure. An acute inflammatory response is triggered at 50 and 25 microg doses by 24 h post-exposure but an inflammatory response is not triggered by a 5 microg dose. Lung inflammation observed at a 50 microg dose at 24 h was resolving by 7d. This work suggests that novel nanostructures with biological design may negate toxicity concerns for biomedical applications of nanotubes. This study also demonstrates that water-soluble rosette nanotube structures represent low pulmonary toxicity, likely due to their biologically inspired design, and their self-assembled architecture.     

杀虫剂对烟粉虱成虫毒力的微量筛选方法

采用直径为18mm的指形管代替常规浸叶法中的培养皿,研究不同寄主植物叶片和琼脂浓度对烟粉虱Bemisia tabaci(Gennadius)成虫自然死亡的影响,并用吡虫啉和啶虫脒为供试药剂对烟粉虱成虫毒力的筛选方法进行比较。结果表明:选用棉花叶片和14g.L-1的琼脂时,指形管内烟粉虱成虫72h自然死亡率最低,只有(5.4±1.5)%;采用此方法就吡虫啉和啶虫脒对烟粉虱成虫的毒力进行测定,测定结果重复性较好,节省药剂用量,无需对烟粉虱成虫麻醉,操作简便,达到微量活体筛选的目标。     

Use of in vivo genetic toxicity data for risk assessment

Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods.     

Defining the limits: Protein aggregation and toxicity in vivo

Abstract

The proper folding of proteins to their functional forms is essential to cellular homeostasis. Perhaps not surprisingly, cells have evolved multiple pathways, some overlapping and others complementary, to resolve mis-folded proteins when they arise, ranging from refolding through the action of molecular chaperones to elimination through regulated proteolytic mechanisms. These protein quality control pathways are sufficient, under normal conditions, to maintain a functioning proteome, but in response to diverse environmental, genetic and/or stochastic events, protein mis-folding exceeds the corrective capacity of these pathways, leading to the accumulation of aggregates and ultimately toxicity. Particularly devastating examples of these effects include certain neurodegenerative diseases, such as Huntington’s Disease, which are associated with the expansion of polyglutamine tracks in proteins. In these cases, protein mis-folding and aggregation are clear contributors to pathogenesis, but uncovering the precise mechanistic links between the two events remains an area of active research. Studies in the yeast Saccharomyces cerevisiae and other model systems have uncovered previously unanticipated complexity in aggregation pathways, the contributions of protein quality control processes to them and the cellular perturbations that result from them. Together these studies suggest that aggregate interactions and localization, rather than their size, are the crucial considerations in understanding the molecular basis of toxicity.     

Possible regulation of the conventional calpain system by skeletal muscle-specific calpain, p94/calpain 3

p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a "modulator protease" in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94:exons 6(-)15(-)16(-) (p94delta), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca(2+)-independent autolytic activity, p94delta was stably expressed in COS and insect cells. p94delta showed Ca(2+)-dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94delta and is a substrate for p94delta, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94delta, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.     

Comparison of calpain I and calpain II from carp muscle

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.