O objetivo deste trabalho foi avaliar o potencial de utilização de marcadores moleculares em genes candidatos da calpaína (CAPN) e calpastatina (CAST) como ferramenta auxiliar para programas de melhoramento de características relacionadas ao crescimento e maciez da carne. Foram avaliados 605 bovinos da raça Nelore, pertencentes à Agropecuária CFM Ltda, com idade média ao abate de 24 meses. Após a extração do DNA de amostras de sangue, por desproteinização em presença de NaCl, a identificação e determinação do polimorfismo para os marcadores moleculares CAPN316, CAPN530, CAPN4751, CAPN4753 e UOGACAST1, foi realizada pelo sistema de detecção TaqManTM utilizando-se PCR em Tempo Real. A análise de maciez da carne, aos 7, 14 e 21 dias de maturação, foi realizada com amostras de carne do Longissimus dorsi, retiradas entre a 12ª e 13ª costela e cisalhadas utilizando-se um Warner Bratzler Shear Force. Nenhum efeito significativo dos marcadores avaliados foi observado para as características de crescimento. Foi verificado efeito significativo, em relação à maciez da carne, para os seguintes polimorfismos: aos 7, 14 e 21 dias de maturação para o marcador CAPN4751; aos 21 dias para o marcador CAPN4753 e aos 14 e 21 dias para o marcador UOGCAST1. Em relação aos efeitos das combinações genotípicas para os marcadores dois a dois, os resultados foram significativos para a combinação CAPN4751/UOGCAST1 nos três tempos de maturação. Para a combinação de marcadores CAPN4753/UOGCAST1 também foram verificados resultados significativos para carnes maturadas aos 14 e 21 dias. Os resultados observados neste trabalho sugerem a possibilidade da utilização de seleção assistida por marcadores (MAS), visando o aumento da qualidade da carne em bovinos da raça Nelore. / The objective of this study was to evaluate the potential utilization of molecular markers on candidate calpain and calpastati n genes as an auxiliary tool for breeding programs on traits related to growth and meat tenderness. A total of 605 Nellore animals, raised by CFM Agro-pecuária Ltda, were used in this study and slaughtered with 24 months in average. After DNA blood samples extraction, by desproteinization in presence of NaCl, the polymorphism (CAPN316, CAPN530, CAPN4751, CAPN4753 and UOGACAST1) identification and determination was realized by TaqManTM detection system using real time PCR. The meat tenderness analysis, at the 7, 14 and 21 days of maturation was realized with Longissimus dorsi meat samples, taken at the 12th and 13th rib interval and Warner Bratzler Peak Shear Force measurements were used. There were no significant effects of molecular markers in growth traits. There were significant effects, regarding to meat tenderness, for following polymorphisms: at 7, 14 and 21 days of maturation, for CAPN4751 marker; at 21 days of maturation, for CAPN4753, and finally, at 14 and 21 days of maturation, for UOGCAST1 marker. In respect to genotypic combination effects analysis for pairwise marker, the results were significant for CAPN4751/UOGCAST1 in three days of maturation. In combination effects for CAPN4753/UOGCAST1 markers, significant effects were also observed for meat tenderness at 14 and 21 days. Theses results suggest that marked selection assisted (MAS) can be used to improve meat quality in Nellore beef cattle.

Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica / d by in vivo micro dialysis. Based on these observations, we conclude that calpain cleavage of fGAD65 occurs under pathological conditions. / by Chandana Buddhala. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Diabetes mellitus represents a group of metabolic disorders caused by both environmental and genetic factors. The two most common forms of diabetes are type 2 diabetes (T2D) and type 1 diabetes (T1D). T2D is associated with obesity and the disease is caused by insulin resistance and pancreatic b-cell dysfunction. T1D is an autoimmune disease in which the insulin- producing b-cells in the pancreas are destroyed by infiltration of lymphocytes. The aim of this thesis was to identify genes conferring susceptibility to diabetes. This was approached using genetic methods, both linkage and association studies, within the population of northern Sweden. The northern Swedish population is well suited for genetic studies of familial forms of disease, since an internal expansion of the northern Swedish population, coupled with a low frequency of immigration and a high frequency of consanguineous marriages, has resulted in a relatively homogeneous gene pool. This simplified genetic background increases the probability of identifying genes contributing to disease. The family-based material used for the type 2 diabetes studies (papers I and II) consisted of 231 individuals from 59 families originating in northern Sweden. The type 2 diabetes case-control material (papers I and II) consisted of 872 cases and 857 matched controls, all from northern Sweden. In paper I we performed a genome-wide linkage scan, seeking T2D susceptibility loci. Linkage to the previously identified Calpain-10 region was found, however, association studies in the case-control material revealed no association to the CAPN10 gene. Using both the family-based and the case-control material, we were able to confirm the association of polymorphisms in the TCF7L2 gene to T2D in the population of northern Sweden (paper II). CTLA-4 is a negative regulator of T cell activity, belonging to the CD28 co-stimulatory receptor family. Numerous reports, including our own, have associated CTLA-4 variants with T1D as well as other autoimmune diseases, such as autoimmune thyroid disease (AITD). Allelic variation in the 3ÚTR of the CTLA-4 gene was associated to human T1D and this variant has also been suggested to affect the level of mRNA encoding the soluble form of the molecule (sCTLA-4). We confirmed the association of allelic variation in the 3ÚTR of the CTLA-4 gene in a T1D/AITD case-control material from northern Sweden, consisting of 104 individuals with ATID, 149 individuals with T1D and 865 matched controls. However, we were unable to identify any correlation between allelic variants in the 3ÚTR of the CTLA-4 gene and expression of sCTLA-4 (paper III). Based on recently published genome-wide association (GWA) scans, 33 single-nucleotide polymorphisms (SNPs) located within 16 genes were selected for an association analysis in T1D/AITD families from northern Sweden. The T1D/AITD family-based material consisted of 253 cases and 206 healthy individuals from 97 northern Swedish families. Analysis revealed association to T1D for SNPs in PTPN22, COL1A2, IL-2Ra and INS. In addition, SNPs in CTLA-4, IL-2 and C12orf30 were shown to be associated to AITD (paper IV). Together, these results underpin the notion that the population of northern Sweden is well suited for the detection of genes involved in complex diseases. The use of our more restricted patient material, compared to materials used in published GWA scans, enables the discovery of disease associated genes in a more cost effective manner and show that our population is capable of detecting general susceptibility genes.

Cysteine proteases are important drug targets due to their involvement in many biological processes such as protein turnover, digestion, blood coagulation, apoptosis, cell differentiation, cell signaling, and the immune response. In this thesis, we have reported the design, synthesis and evaluation of clan CA and clan CD cysteine protease inhibitors. Aza-peptidyl Michael acceptor and epoxide inhibitors for asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE) and the hard tick, Ixodes ricinus (IrAE) were designed and synthesized. SARs were similar, but with some notable exceptions. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased inhibitory potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues in the P2 position. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors and, for some of these compounds, second order inhibition rate constants are the fastest yet discovered. We have also synthesized aza-peptidyl Michael acceptor and epoxide inhibitors for the parasitic cysteine proteases; cruzain, rhodesain. We have found that monosubstituted amides were favored over disubstituted amides indicating the involvement of the amide hydrogen in a H-bond network. We have shown that aza-peptide epoxides were as potent as Michael acceptors and we have obtained compounds with IC50 values as low as 20 nM. We have worked on the synthesis of heterocyclic peptidyl α-ketoamides, peptidyl ketones and aza-peptidyl ketones as calpain inhibitors. We have synthesized peptidyl α-ketoamides with nucleotide bases in the primed region to create compounds that can cross the blood-brain barrier. We have improved the potency by introducing a hydrophobic group on the adenine ring. We have obtained compounds with Ki values in the nanomolar range. We have designed peptidyl aminoketones as a new class of inhibitors for calpain. Peptidyl aminoketones were less potent than peptidyl α-ketoamides but still reasonable inhibitors of calpain that have the potential to cross the BBB.

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

Biochemical changes in muscle postmortem have been associated with initial beef tenderness early postmortem, and with improvements in tenderness during postmortem storage, defined as meat aging. Differences in the initial contractile state of the sarcomere, the ionic environment of the sarcoplasm including pH, the activity of neutral proteolytic enzymes, and collagen content and solubility have been associated with beef tenderness. In Phase I, steaks from four genetic lines of steers and heifers were used to understand the biochemical differences between tough and tender steaks. The most tender (< 30 N Warner Bratzler shear force (WBS)) and toughest Longissimus steaks (< 30 N WBS) from Angus, Braford, Brangus, and Simbrah heifers and steers were used. For Phase II, samples were obtained from a subset of Santa Cruz yearling heifers selected based of genotypes for tenderness (tough and tender) using a commercial genetic marker. Within genotype for tenderness, each animal was randomly assigned to one of four growth enhancement treatments. The most tender (< 30 N WBS) and toughest Longissimus steaks (< 30 N WBS) were selected for use in this study. In Phase I, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0005) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P = 0.008) marbling score and (P = 0.01) Quality grade. Sarcomere length, total and soluble collagen, potassium concentration, and m and µcalpain did not differ (P > 0.05) between tough and tender steaks. Sodium concentration at 10 d was higher (P = 0.03) in tough steaks, but only account for 0.05% of the variation in WBS at 3d. Tender steaks had less (P = 0.04) intact desmin at 24h, but intact desmin was not correlated (P > 0.05) with WBS. In Phase II, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0001) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P < 0.03) marbling score and (P = 0.02) Quality grade. Tender teaks were slightly lighter (P = 0.02), with more red (P = 0.02) and yellow (P = 0.007) color, and had slightly lower (P = 0.02) pH, compared with tough steaks. Sarcomere length, total and soluble collagen, sodium and potassium concentration, and m and µcalpain did not differ (P > 0.05) between tough and tender steaks. Tender steaks had less (P < 0.0001) intact desmin at 17d postmortem than tough steaks. Intact desmin at 17d was responsible for 4%, 47%, and 30% of WBS variation after 3, 10, and 17d postmortem, respectively. The slight difference in marbling and quality grade did not account for a significant amount of variation in WBS. However, meat color and pH accounted for variation in shear WBS. Calcium flux may have influenced meat tenderness by activation of calpains and may have altered protein to protein interactions. Results suggested that marbling, µ calpain activity, and desmin degradation, and to a lesser extent pH and meat color contributed to meat tenderness.

The calpain family of intracellular Ca2+-dependent cysteine proteases is involved in a number of intracellular signaling processes. Calpain hyperactivity has also been implicated in ischemic injury, neurodegenerative diseases and cataract formation. However, the specific function of calpains in these normal and diseased states remains unclear. Competitive inhibition of calpain is useful for studying their functions and can lead to pharmacological treatments, while monitoring their activity with activity-based probes (ABPs) can reveal how calpain is regulated and be applied to screen for inhibitors in vivo. But these strategies are complicated by the similarity of the calpain active-site when compared to other intracellular cysteine proteases. Therefore, there is a need to design inhibitors and ABPs that selectively target calpain. Using X-ray crystallography, the interactions between the calpain active-site and each of two reversible inhibitors was studied. This led to the discovery of novel non-covalent aromatic stacking and hydrogen bonding interactions between the primed-side adenine group of one inhibitor and indole ring of an active-site Trp residue in μ-calpain. A substrate-based competition assay later confirmed that these interactions provided this compound with an inhibitory advantage over the other, which lacked any primed-side interactions, thereby providing insight into the development of new, more specific reversible calpain inhibitors. Next, a fluorescent ABP, containing features borrowed from an irreversible and presumably calpain-specific inhibitor, was evaluated for its ability to detect calpain activitiy. Although this probe appropriately targeted the calpain active site in its Ca2+-activated form, it was unable to detect calpain activity in a cell extract. Nevertheless, the results of this study have yielded insights into ways of improving the calpain detecting ability of this ABP. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-01 15:39:07.023

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

No description available.

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and depression in sarcolemmal Na+-K+-ATPase activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, and reperfusion after 45 min of ischemia. Decreases in Na+-K+-ATPase activity at 60 min of global ischemia were associated with augmented activities of both calpain and MMPs and depressed protein content of β1- and β2-subunits, without changes in α1- and α2-subunits of the enzyme. However, reperfusion of ischemic heart produced depression in Na+-K+-ATPase activity, no change in the augmented calpain activity, but decreases in augmented MMP-2 activity and Na+-K+-ATPase content. MDL28170, a calpain inhibitor, was more effective in attenuating I/R-induced alterations than doxycycline, an MMP inhibitor. Incubation of control SL preparation with calpain, unlike MMP-2, depressed Na+-K+-ATPase activity and decreased α1, α2 and β2 without changes in β1. These results support the view that activation of calpain is involved in depressing Na+-K+-ATPase activity and degradation of its subunits in hearts subjected to I/R injury.