Dissertations / Theses on the topic 'Calpain 2'

Introdução: A hipertrofia cardíaca induzida por sobrecarga hemodinâmica crônica (HC) é caracterizada por espessamento das paredes do ventrículo esquerdo e do tecido intersticial. As atividades aumentadas de calpaína-1 e metaloproteinase de matriz(MMP)-2 são observadas em diferentes modelos de hipertensão arterial e estão relacionadas com as mudanças fisiopatológicas na HC. Por outro lado, a atividade de MMP-2 parece ser modulada positivamente por ativação de calpaína-1 em diferentes modelos. O objetivo deste trabalho é analizar se a calpaína-1 contribui para o aumento da atividade de MMP-2 no coração e se esse mecanismo resulta nas mudanças crônicas cardíacas na hipertensão renovascular. Métodos: Ratos Wistar submetidos ao modelo de 2-rins-1 clipe (2R-1C)(180-200g) e seus respectivos controles (Sham) foram tratados com verapamil (VRP), um bloqueador de canais para cálcio tipo L (BCCL, 8mg/kg/bid) ou veículo durante 8 semanas. O BCCL reduz as concentrações intracelulares de cálcio, o que leva à diminuição da ativação de calpaína-1, e então à possível modulação da atividade e expressão proteica de MMP-2. Pressão arterial sistólica (PAS) dos ratos foi monitorada durante 10 semanas de hipertensão por pletismografia de cauda. O ventrículo esquerdo (VE) foi analisado por histologia e ecocardiografia para avaliação das dimensões ventriculares. A atividade de calpaína-1 e MMP-2 foi avaliada por zimografia em gel. A expessão proteica de calpaína-1 e MMP-2 foi avaliada por western blot e imunofluorescência. Os corações foram submetidos à avaliação funcional por Langendorff. Todos os protocolos foram aprovados pelo Comitê de Ética em Pesquisa Animal da Faculdade de Medicina de Ribeirão Preto (43/2017). Resultados: Após 10 semanas, a PAS teve um aumento sustentado nos animais 2R-1C e o tratamento com VRP não foi capaz de reduzí-la em nenhum tempo de hipertensão. O peso corporal não apresentou diferença significativa entre os grupos. O grupo hipertenso teve um aumento da massa cardíaca quando comparado ao sham e o tratamento com verapamil reduziu esse parâmetro. A análise da espessura do ventrículo esquerdo demonstra que o VRP é capaz de reverter a HC induzida por sobrecarga pressórica nos animais hipertensos. Os animais 2R-1C apresentaram um aumento singificativo na expressão proteica e atividade de calpaína-1 e o VRP foi capaz de diminuir esses níveis. Foi observado aumento da atividade das isoformas de MMP-2 nos ratos 2R-1C quando comparados aos controles e o VRP foi capaz de reduzir a atividade da isoforma de 64kDa. A contratilidade cardíaca intrínseca dos animais 2R-1C sugere uma disfunção cardíaca quando comparados aos controles sham, embora a fração de ejeção desses animais esteja preservada. O VRP não foi capaz de alterar esses parâmetros. Conclusão: O VRP pode contribuir para a redução da hipertrofia cardíaca por diminuir a expressão proteica de calpaína-1 e MMP-2 na hipertensão renovascular. Apoio financeiro: Capes, CNPq, FAPESP
Introduction: The chronic hemodynamic overload-induced cardiac hypertrophy (CH) is characterized by thickening of the left ventricle walls and hypertrophy of the cardiomyocytes and interstitial tissue. Increased activity of calpain-1 and matrix metalloproteinase(MMP)-2 was observed in different models of arterial hypertension models and contributes to the pathophysiologic changes shown in CH. On the other hand, MMP-2 activity is also positively modulated by activation of calpain-1 in different animal models of cardiovascular diseases. The objectives here are to analyze whether calpain-1 contributes to increase the activity of MMP-2 in the heart and whether this mechanism results in chronic cardiac changes in the renovascular hypertension. Methods: Two kidney-one clip (2K1C) hypertensive male Wistar rats (180-200g) and their respective controls (Sham) were orally treated with verapamil (VRP), a L-type calcium channels blocker (LCCB, 8mg/kg/bid), or vehicle during 8 weeks. The LCCB reduces the intracellular concentration of calcium, thus decreasing the activation of calpain-1, and then may modulate the activity of MMP-2. Systolic blood pressure (SBP) was monitored in the rats during 10 weeks of hypertension. Left ventricle (LV) was analyzed by histology and echocardiography to evaluate ventricle thickening. Calpain- 1 and MMP-2 activities were analyzed by zymography and their expression by immunofluorescence and western blot. Hearts were submitted to functional evaluation by Langendorff. All the protocols were approved by the Ethical Committee in Animal Research of Ribeirao Preto Medical School (43/2017). Results: After 10 weeks, the systolic blood pressure had sustained increase and treatment with VRP was not able to decrease it in any time of hypertension. The body weight did not present significant changes between the groups. Hypertensive group had significant increase in the ventricle/body weight ratio (VW/BW) when compared to sham and treatment with VRP decreased it. Analysis of ventricle thickening showed that VRP is able to revert CHinduced pressure overload. The 2K-1C rats showed a significant increase in the activity and expression of calpain-1 in the heart and VRP reverted it. It was also observed increased activity of MMP-2 forms in the hypertensive rats and VRP decreased the 64kDa MMP-2 activity. The 2K-1C group had cardiac dysfunction when compared to controls groups, and VRP did not alter it. The ejection fraction was not changed in 2K- 1C rats. Conclusion: VRP decreased expression and activity of calpain-1 and MMP-2 in the hearts of 2K-1C rats and then contributed to ameliorate hypertension-induced cardiac hypertrophy

Dengue virus (DENV) is an arthropod-borne virus and has become a worldwide problem with steadily rising annual infection rates. Patients present with a range of symptoms from mild fever to, in some cases, life-threatening hemorrhagic fever and shock syndrome. The most severe cases require emergency hospital care and currently, there is no effective drug treatment or vaccine for dengue. As severe symptoms appear post-peak viremia, immuno-pathology is thought to be the cause and a potential trigger of this is differential activation of the immune response upon recognition of DENV. This could be due to a combination of factors including varying receptors, signalling pathways and immune regulation mechanisms. In order to understand DENV infection better, it is imperative to study the mechanisms of activation and control of immune responses triggered by the virus. Very early events in viral infection (after 10 min stimulation) were studied aiming to identify proteins involved in differential activation of immune responses. Phosphorylated proteins were isolated from cells post-stimulation and analysed by mass spectrometry. More than 200 proteins were differentially regulated by phosphorylation in response to DENV stimulation as compared to Mock, Influenza A virus and LPS stimulation. The effect of two specific proteins, namely Calpain-2 and Importin-5, identified to be differentially phosphorylated was investigated further. Calpain-2 was seen to be vital in the efficient production of progeny virions and the transcription of Mx1, an anti-viral interferon stimulated gene. Importin-5 is known to transport DENV NS5 into the nucleus during infection and was seen to co-precipitate with many host proteins. In summary, it is imperative that novel treatments and vaccines are developed for dengue as it is one of the world’s most prevalent arthropod-borne viruses. It was discovered here that many proteins undergo phosphorylation/de-phosphorylation in response to DENV stimulation to a differing degree than other stimuli. Calpain-2 plays a vital role DENV infection, potentially influencing the potency of immune response. Importin-5 associates with various host proteins during DENV infection, potentially altering their function or the function of Importin-5 itself. Research into targeted inhibition of Calpain-2 function or Importin-5 interaction with DENV NS5 could lead to a successful anti-viral treatment for DENV infection.

La spasticité et la douleur neuropathique sont deux symptômes apparaissant fréquemment après une lésion médullaire. La spasticité est définie comme une augmentation du tonus musculaire qui provoque des contractures, tandis que la douleur neuropathique se caractérise par des sensations douloureuses survenant suite à une lésion du système nerveux.Ces deux symptômes résultent en partie d’une désinhibition des réseaux neuronaux sous-lésionnels lié à une diminution de l’expression du cotransporteur potassium-chlorure type 2 (KCC2). Pour être efficace,l’inhibition nécessite l’action de cette protéine qui extrait les ions chlorure des neurones.L’objectif de la présente thèse est donc d’identifier des médicaments capables d’activer KCC2 afin de restaurer l’inhibition dans le but de traiter la spasticité et la douleur neuropathique.Dans un premier temps, nos résultats ont montré que l’activation de récepteurs sérotoninergiques 5-HT2A avec le TCB-2 rétablit l’expression de KCC2 dans la corne dorsale après une lésion médullaire ou névrectomie. Or le TCB-2 réduit seulement la douleur neuropathique après la lésion spinale.Par la suite, nous avons identifié la prochlorperazine comme une molécule augmentant l’activité de KCC2. Si la prochlorperazine est efficace contre la spasticité, elle a néanmoins un effet plus modeste envers l’allodynie mécanique suite à une lésion médullaire.Enfin, nous avons démontré que la diminution de KCC2,ainsi que l’hyperexcitabilité des motoneurones suite à la lésion, dépendent de l’activation des calpaïnes.Cette thèse valide KCC2 comme une cible thérapeutique dans le traitement de la spasticité et la douleur neuropathique suite à une lésion médullaire
Spasticity and neuropathic pain are two symptoms that arise frequently after a spinal cord injury. Spasticity is defined as an increase of the muscle tone contributing to cramps, whereas neuropathic pain consists of painful responses caused by a damaged nervous system. Both symptoms arise, in part, due to a loss of inhibition in the sublesional neural networks, linked to a downregulation of the expression of potassium-chloride cotransporter type 2 (KCC2). For inhibition to be efficient, the action of this protein, which extrudes chloride ions from neurons, is needed.The objective of this thesis is, therefore, to identify drugs capable of activating KCC2 to recover inhibition with the objective of treating spasticity and neuropathic pain.First, our results have proven that the activation of serotonin receptors 5-HT2A with TCB-2 restores KCC2 expression in the dorsal horn after a spinal cord or peripheral nerve injury. However, TCB-2 reduces neuropathic pain after a spinal cord injury exclusively.In the next stage of the work, we have identified prochlorperazine as an enhancer of KCC2 activity. Prochlorperazine is efficient against spasticity, although it only showed a modest reduction of mechanical hyperalgesia in animals with a spinal cord injury.Lastly, we have proven that KCC2 downregulation and motoneuron hyperexcitability after a spinal cord injury depend on the overactivation of calpains.This thesis validates KCC2 as a druggable target to treat spasticity and neuropathic pain after spinal cord injury

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

La calpaïne 1 est une protéase à cystéine activée par le calcium, qui peut être partiellement externalisée. Les calpaines extracellulaires favorisent la résolution de l'inflammation et la réparation des tissus, à travers la prolifération et la migration cellulaire. Le récepteur Toll like (TLR) 2 a été identifié comme une cible des calpaïnes extracellulaires dans les lymphocytes. L'objectif est d'étudier le rôle de la calpaïne extracellulaire 1 dans la progression tumorale de l'adénocarcinome pulmonaire lepidique (ADL). La calpaïne extracellulaire, le fragment soluble de TLR2, le versican et les cytokines étaient analysés par ELISA dans des surnageants de lavage bronchoalvéolaire (LBA) de patients atteints d'ADL (n = 68). La source de calpaïne était analysée par immunohistochimie. TLR2, cible de la calpaïne extracellulaire était étudiée par cytométrie de flux sur les polynucléaires neutrophiles (PNN) et des lignées humaines de cancer bronchiques. Calpain 1 extracellulaire, sécrété par les cellules tumorales, était associée à la progression tumorale, l'inflammation à neutrophiles, avec un facteur de mauvais pronostic de survie (p = 0,003). TLR2 était exprimé sur les cellules tumorales ou les PNN avec une diminution d¿expression après traitement par calpaïne. Le fragment soluble de TLR2 était corrélée à la concentration extracellulaire de calpaïne 1 dans les surnageants de LBA (r = 0,624; p <0,001). Le fragment soluble de TLR2 élevé était associé à la progression tumorale et un environnement pro-inflammatoire La calpain extracellulaire sécrétée par la cellule tumorale, favorise un microenvironnement inflammatoire et la progression tumorale médiée par TLR2 dans ADL
Calpain 1 is pro inflammatory calcium-activated cysteine proteases, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the role of extracellular calpain 1 in tumor progression of lepidic pulmonary adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry. TLR2 as target of extracellular calpain was studied by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular Calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (p=0,003). TLR2 was expressed on PMN or tumor cells and decreased after calpain treatment. The soluble fragment of TLR2 was correlated to the extracellular calpain 1 concentration in the BALF supernatants (r=0.624; p<0.001). High soluble fragment of TLR2 was associated with tumor progression and a pro-inflammatory environment. Extracellular Calpain 1 secreted by tumor cell, promotes inflammatory microenvironment and tumor progression through TLR2 in LPA

Calpains are a family of intracellular cysteine proteases activated by calcium. They participate in many processes including cell motility, cell cycle progression and cell death, in response to calcium signaling. Because calpain over-activation as a result of calcium dysregulation is a contributing factor to many disease states, these enzymes are important therapeutic targets. Within the cell, calpains 1 and 2 are regulated by the protein inhibitor calpastatin. This unstructured protein is specific for calpain, binds tightly, and recognizes only the activated form of the enzyme. Detailed kinetic data obtained using surface plasmon resonance allowed the association and dissociation rates of each of the four calpastatin inhibitory domains to be measured. Based on this, inhibitory domain 4 was selected to be co-crystallized bound to calpain 2. The X-ray crystal structure of this complex provided both the first view of the active enzyme, as well as the first view of how it is inhibited. Calpastatin wraps around the enzyme making contact with each domain. It lies in the active site as a contiguous polypeptide chain and escapes cleavage by forming a loop away from the catalytic cysteine. In addition to inhibiting substrate cleavage, calpastatin protects calpain in two ways; it prevents autoproteolysis, and it prevents calcium-dependent aggregation. The crystal structure of the calpastatin:calpain complex revealed no obvious reason for this stabilization. To elucidate how this protection occurs, peptides were synthesized corresponding to conserved subdomains of calpastatin. Surprisingly, each peptide alone was capable of preventing aggregation in vitro, by blocking hydrophobic patches exposed upon activation. The increased hydrophobic surface of the activated enzyme may alter calpain’s affinity for other proteins such as substrates. By binding across many domains of calpain, calpastatin could act to block protein-protein interactions. These studies have characterized calpastatin’s interaction with calpain, which will further our understanding of the enzyme’s regulation and aid in the development of better calpain inhibitors.
Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-04-29 15:27:16.208

Glioblastoma is the most malignant primary brain tumor with the average patients surviving only one year after diagnosis, even with aggressive therapy. The formation of numerous micro-tumors dispersed into the brain due to rapid invasion of tumor cells, presents the primary challenge to the surgical removal of tumors and limits the effectiveness of current treatments. This dissertation presents studies aimed at understanding the molecular mechanisms regulating invasion of human glioblastoma cells. Transplantation of human glioblastoma cells in the zebrafish brain showed that the knockdown of calpain 2, a calcium-activated protease, resulted in a three fold decrease in the tumor cell invasion. The result was further verified in the organotypic mouse brain slices where the knockdown cells demonstrated 2-fold decrease in the area of dispersal compared to control cells. Our data show that calpain 2 plays a role in the process of tumor cell angiogenesis. Glioblastoma cells were transplanted into the brain of zebrafish expressing GFP in the blood vessels and we observed that 23% of animals injected with control tumor cells demonstrated angiogenesis. In contrast, only 9% of fish that received calpain 2 knockdown cells showed the formation of new vessels. Consistent to the reports from human glioblastoma patients and rodent models, we did not observe metastasis of transplanted cells outside of the brain in the zebrafish, supporting for the use of zebrafish as an important model for glioblastoma cell invasion studies. These results provide evidence that calpain 2 protease activity is required for the dispersal of glioblastoma cells in the brain microenvironment. To determine the mechanism of calpain 2 regulation of tumor cell invasion, proteolysis of filamin by calpain 2 was studied. Filamin is an important actin cross-linking protein which develops orthogonal actin networks in the periphery of the cell. In this study, we show that the expression of filamin inhibits glioblastoma cell invasion. Hence, knocking down filamin expression by 80% resulted in 220% increase in the invasion of glioblastoma cells through Matrigel extracellular matrix. The regulated proteolysis of filamin is a potential mechanism to facilitate the cyclic turnover of actin orthogonal networks which is required for glioblastoma cell invasion. In this study, we identified a novel mechanism that the PI3 kinase activity regulates the cleavage of filamin by calpain 2 in glioblastoma cells. Binding of a membrane phospholipid phosphatidylinositol (3,4,5) triphosphate [PtdIns (3,4,5)-P₃] to filamin induces its proteolysis by calpain 2 after the amino acid lysine 268, removing the actin binding domain which in-turn abolishes the actin binding ability of filamin.
Graduation date: 2011
Access restricted to the OSU Community at author's request from Jan. 31, 2011 - Jan. 31, 2012

Calpains are calcium-dependent, intracellular, multi-domain cysteine proteases involved in many physiological functions regulated by calcium signaling, including cell motility. How calpains are activated in the cell is still unknown because the resting intracellular concentration of Ca2+ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Several stratagems by which calpains might overcome this Ca2+ concentration differential have been proposed. It is possible that post-translational modifications like phosphorylation, or accessory proteins that bind to calpain, might facilitate the enzyme’s activation at lower than optimal Ca2+ concentrations. Autoproteolysis (autolysis) and subunit dissociation are two other proposed activation mechanisms that could release constraints on the calpain core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, it was demonstrated that while the anchor helix is one of the first targets to be cut, several other potentially inactivating autolysis sites, particularly in Domain III, can also be cleaved within the first minute. Thus autolytic activation would go hand in hand with inactivation. By fractionating and identifying calpain 2 autolysis fragments, I show that the small subunit does not dissociate away from the large subunit, but is proteolyzed to a 40-45 k heterodimer of the penta-EF-hand Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit domain VI homodimer that would be produced by subunit dissociation. A calpastatin affinity column was constructed and used to capture recombinant calpain 2 from bacterial cell lysate. This affinity column provides a tool to screen for and capture calpain complexed to potential binding partners in the presence of Ca2+. Here I propose a model for calpain 2 activation in vitro that does not involve autolysis, subunit dissociation, or calpain activators.
Thesis (Master, Biochemistry) -- Queen's University, 2010-10-25 16:03:52.364

博士
國防醫學院
生命科學研究所
97
The calpain family of proteases are important for several key aspects of cell motility, including cell spreading, membrane protrusion, detachment of the rear and integrin, growth-factor-mediated signaling. The calpain activity can be activated by calcium, phospholipid binding, autolysis, serine phosphorylation and inhibited by calpastatin. In this study, we have explored two post-translational modifications, tyrosine phosphorylation and sumoylation, involved in regulating calpain-2 activity. Endothelial/epithelial tyrosine kinase (Etk) increases calpain-2 activity through direct tyrosine phosphorylation both in vitro and in vivo at Tyr-146. Phosphorylation of Tyr-146 is required for epidermal growth factor (EGF)-induced calpain-2 activation. Moreover, the phosphorylation of calpain-2 Y146 is also important for vascular endothelial growth factor (VEGF)-induced endothelial cell migration and angiogenesis. More importantly, Etk mediates both EGF and VEGF-induced calpain-2 Y146 phosphorylation. Converting Y146 to phenylalanine limits EGF and VEGF-mediated calpain-2 activation and cell deadhesion and migration, respectively. Overexpression of Y146F by recombinant adenovirus blocked VEGF-induced angiogenesis. Altogether, these findings strongly suggest that Etk-mediated calpain activation via Y146 phosphorylation is crucial for EGF and VEGF-induced cell deadhesion and cell migration. In addition, we also found that calpain-2 can be SUMO modified at lysine residue 390. Converting the SUMO acceptor lysine residue to arginine residue significantly attenuated calpain-2 activity, correlating well with a loss of calpain-2-elicited cell motility. Accordingly, expression of SENP1 could abrogate calpain-2 sumoylation, causing an inhibition on calpain-2-dependent activity and cell motility. These results not only identify calpain-2 as a substrate for sumoylation but also provide an important role of sumoylation in regulating cell migration. Taken together, these data provide evidence that calpain-2 can be activated through tyrosine phosphorylation and sumoylation.

碩士
國防醫學院
生物化學研究所
97
Mouse alpha actinin 2 (mACTN2) belongs to the spectrin protein superfamily, which is composed of 894 residues and expresses in the heart, skeletal muscle, lung, and brain. Our previous study demonstrated that mACTN2 and its superfamily member contain LXXLL motif, the Nuclear receptor (NR) binding motif, and serve as a coactivator for nuclear receptor, mACTN2 also bind to the C-terminal region of GRIP1 (glucocorticoid receptor interacting protein 1) for the secondary nuclear receptor functions. Our previous study identified the NES (nuclear export signals) of mACTN2 for the regulation of its nucleo-cytoplasmic trafficking which was correlated with its protein stability in the cytoplasmic subcellular localization. The recent research suggested calpain could cleavage alpha actinin. In this thesis, I further examined calpain whether was able to affect nuclear or cytolasmic mACTN2 protein stability. My data demonstrated that calpain primarily cleavaged cytoplasmic mACTN2 and then decreased its protein stability via non-conserved calpain cleavage sequence. In addition to the calpain inhibitor, the stabilization of cytoplasmic mACTN2 protein, mediated through the decrease of endogenous calpain to suppress that rate of cleavage. Finally, the differential role of nuclear localization sequence and NES in the mACTN2 might determine its subcellular localization, protein stability, and subsequent endogenous functions.

Type 2 diabetes is characterized by an inability to maintain normoglycemia due to impairments in both insulin secretion and action. The impaired capacity of the pancreatic beta cell to secrete sufficient concentrations of insulin directly contributes to the worsening glycemic control. High concentrations of glucose and free fatty acids have been shown to be deleterious for pancreatic beta cell function and viability. Increased availability of glucose and free fatty acids may also contribute to the aggregation of islet amyloid polypeptide (IAPP) and formation of islet amyloid, a commonly observed pathology in type 2 diabetes associated with decreased beta cell viability. The mechanism(s) mediating the continuum of declining pancreatic beta cell function towards increased pancreatic beta cell death under conditions of increased glucose and free fatty acid availability are not completely understood. Insight into the pathways contributing to pancreatic beta cell dysfunction and death could provide avenues for therapeutic strategies aimed at improving glycemic control in type 2 diabetes. In this thesis, the role of the calpain and caspase families in mediating the toxic effects of IAPP, glucose and free fatty acids in the pathogenesis of impaired beta cell function and viability were assessed. The propensity of IAPP to aggregate in vitro was greatly influenced by an increased availability of the peptide and a neutral pH, replicative of conditions observed during increased glucose and free fatty acid availability. The toxicity of the IAPP aggregates was not found to be solely dependent upon the activation of either the caspase or calpain family. However, culture of the pancreatic beta cell line INS-1 in elevated glucose concentrations was found to increase secretory vesicle release under nonstimulatory conditions in a caspase-dependent manner. Additionally, fatty acid-induced pancreatic beta cell death was found to be dependent upon activation of the caspase but not the calpain family and was evident only at increased glucose concentrations. These results implicate a role for the caspase family in mediating the deleterious effects of increased glucose and free fatty acid availability upon beta cell function and viability. The beta cell function and viability in patients with type 2 diabetes may potentially benefit from strategies aimed at inhibiting caspase activity or reducing free fatty availability.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

博士
國防醫學院
生命科學研究所
101
Long-term memory requires the activity-dependent reorganization of the synaptic proteome to modulate synaptic efficacy and consequently consolidate memory. Activity-regulated RNA translation can change the protein composition at the stimulated synapse. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein that represses translation of its target mRNAs in neurons, while activation of N-methyl-D-aspartic acid (NMDA) receptors alleviates this repression. Although recent research has revealed the mechanism of CPEB3-inhibited translation, how NMDA receptor signaling modulates the translational activity of CPEB3 remains unclear. This study shows that the repressor CPEB3 is degraded in NMDA-stimulated neurons and that the degradation of CPEB3 is accompanied by the elevated expression of CPEB3's target, epidermal growth factor receptor (EGFR), mostly at the translational level. Using pharmacological and knockdown approaches, we have identified that calpain 2, activated by the influx of calcium through NMDA receptors, proteolyzes the N-terminal repression motif but not the C-terminal RNA-binding domain of CPEB3. As a result, the calpain 2-cleaved CPEB3 fragment binds to RNA but fails to repress translation. Therefore, the cleavage of CPEB3 by NMDA-activated calpain 2 accounts for the activity-related translation of CPEB3-targeted RNAs.

α-actinin-4 is a non-muscle isoform of α-actinin that belongs to the spectrin superfamily. It comprises three functional regions: an N-terminal actin-binding region that consists of two calponin homology (CH) domains, a central region that consists of four copies of the spectrin-like repeat domain and a C-terminal calmodulin-like domain that is predicted to bind Ca²⁺. α-actinin-4 is organised as an antiparallel homodimer formed by the interaction of four spectrin-like repeats between the two monomers, giving a rod-like shape, with actin binding regions at both ends. α-actinin-4 is an abundant actin-bundling protein, which provides a direct link between actin filaments and integrins, and is believed to play an important role in stabilising cell shape and adhesion and regulating cell migration. It also acts as a tumor suppressor and influences the metastatic potential and invasiveness in human cancers. A cluster of three actin binding motifs have been identified in the CH domains (2X CH) from other members of the spectrin superfamily, utrophin and dystrophin. Two of them reside in the CH1 domain and the third resides in the first α-helix of the CH2 domain. In addition, a PIP2 binding site has been mapped on a region adjacent to actin-binding site-3. These observations imply the F-actin binding activity would be regulated by phosphoinositides. Five mutations of α-actinin-4, K122N, an alternative splice variant, K255E, T259I and S263P, have been reported to be involved in three human diseases, non-small lung cancer (NSCLC), small cell lung cancer (SCLC) and focal segmental glomerulosclerosis (FSGS). The mutation site within these mutants is located on the actin binding region. Therefore, the actin binding region is presumed to be associated with the progression of human disease. The aims of this thesis focused on the regulation of the F-actin binding activity of α-actinin-4 by phosphoinositides (PIP2 and PIP3), the calmodulin-like domain and Ca²⁺ , determination of the three-dimensional structure of the CH2 domain in solution and identification of the phosphoinositide binding site on the CH2 domain. In order to investigate the F-actin binding activity quantitatively, a novel in vitro F-actin binding assay (solid phase) was established to replace the semi-quantitative actin bundling assay. Using this novel solid phase F-actin binding assay, Ca²⁺ was shown to enhance the F-actin binding activity of α-actinin-4 in a concentration-dependent manner. The presence of 10 mM Ca²⁺ results in a two-fold increase in the F-actin binding activity. Both PIP2 and PIP3 inhibited the F-actin-binding activity of α-actinin-4 in a concentration-dependent manner with an approximate IC₅₀ of 75 and 45 μM, respectively. In order to characterise how phosphoinositides regulated the F-actin binding activity of α-actinin-4, the solution structure of α-actinin-4 CH2 domain was determined and the phosphoinositide binding residues within the CH2 domain were identified using NMR spectroscopy. The solution structure of α-actinin-4 CH2 domain contained six α-helices and was similar to that of other spectrin superfamily members. The strategy used in identification of the phosphoinositide binding site was an NMR-based 2D ¹H-¹⁵N HSQC ligand titration assay to replace the traditional semi-quantitative protein-lipid overlay assay. Using the NMR-based ligand titration assay, the recognition site for the inositol head group resides in residues Trp 172, Tyr 265 and His 266 and the binding region of acyl chains resides in the first α-helix structure which is one of the putative F-actin binding sites. In order to examine the interaction of phosphoinositides with this site, Y265A and H266E mutants of α-actinin-4 CH2 domain were generated using site-directed mutagenesis and verified the interaction with phosphoinositides and the inositol head group using an NMR-based ligand titration assay. These results confirmed the phosphoinositide binding site on the CH2 domain and residues, Tyr 265 and His 266, are critical for interacting with phosphoinositides. Wildtype and mutants (Y265A and H266E) of α-actinin-4 were expressed in mammalian cells as EGFP-fusion proteins. Wildtype α-actinin-4 was shown to be co-localised with focal adhesions and actin stress fibres. However, Y265A and H266E mutants of α-actinin-4 were co-localised with actin stress fibres but poorly co-localised with focal adhesions. Moreover, both Y265A and H266E mutants of α-actinin-4 were co-localised with actin in the cytoplasm rather than localised along the cell membrane after EGF stimulation for 30 minutes. These results suggested that PIP2 assists the co-localisation of α-actinin-4 with focal adhesions. Taken together, the results described in this thesis concluded that Ca²⁺ enhanced the F-actin binding activity of α-actinin-4 in vitro. However, phosphoinositides (PIP2 or PIP3) inhibited the F-actin binding activity in vitro. Moreover, the results described in this thesis provided a phosphoinositide binding site on α-actinin-4 CH2 domain. Binding to PIP2 is important to the localisation of α-actinin-4 in focal adhesions.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008