Degradation Of Cytoskeletal Proteins Research Articles

Effects and mechanism of sub-freezing storage on water holding capacity and tenderness of beef

In order to explore the effect of sub-freezing storage on water holding capacity and tenderness of beef, four treatments were compared in this study: sub-freezing (−7 °C) fast sub-freezing (−38 °C until the core temperature achieved to −7 °C), superchilling (−1 °C) and fast frozen (−38 °C until the core temperature achieved to −18 °C) with the latter two treatments serving as the controls. The differences in muscle fiber structure, water distribution, protein oxidation and cytoskeletal protein degradation were studied. The results demonstrated that compared with other treatments, the fast sub-freezing treatment resulted in less structural damage to the muscle fibers and had better water holding capacity. Both sub-freezing and fast sub-freezing treatments inhibited protein oxidation compared with superchilling, but the former treatment's level of protein oxidation was higher than that in fast sub-freezing treatment during long-term storage (42 weeks). In addition, the structural proteins in the sub-freezing and fast sub-freezing treatments underwent faster degradation during long-term storage and therefore the meat was more tender compared with the fast frozen treatment. The results indicate that the fast sub-freezing treatment can be potentially applied in beef storage.

Aging mechanism for improving the tenderness and taste characteristics of meat.

Tenderness and taste characteristics of meat are the key determinants of the meat choices of consumers. This review summarizes the contemporary research on the molecular mechanisms by which postmortem aging of meat improves the tenderness and taste characteristics. The fundamental mechanism by which postmortem aging improves the tenderness of meat involves the operation of the calpain system due to apoptosis, resulting in proteolytic enzyme-induced degradation of cytoskeletal myofibrillar proteins. The improvement of taste characteristics by postmortem aging is mainly explained by the increase in the content of taste-related peptides, free amino acids, and nucleotides produced by increased hydrolysis activity. This review improves our understanding of the published research on tenderness and taste characteristics of meat and provides insights to improve these attributes of meat through postmortem aging.

Влияние сухого созревания на белки мышечной ткани говядины

Meat is an inherent part of human diet. Its quality develops at different stages of production, storage, and processing. In this respect, the stage of aging is especially important. This technology makes it possible to regulate biochemical processes in meat raw materials. Long-term dry aging is a promising method that presupposes conditions that limit the growth of microorganisms. The transformations in the protein component are an important but understudied aspect of meat quality formation during dry aging.
 The research featured Hereford beef carcasses of Siberian breeding. The samples were isolated from the inner part of bone spinal-lumbar cuts after 21, 35, and 42 days of dry aging under the following conditions: 0–1°C, 74–75% relative humidity, 0.5 m/s air velocity. The samples were subjected to dry aging after 24 h at 4°C. The fractional composition of proteins was controlled by vertical electrophoresis in a Mini-Protean Tetra System chamber. The amino acid composition was defined by high-performance liquid chromatography in a Shimadzu LC-20 Prominence liquid chromatograph with a Shimadzu SPD20MA diode-matrix detector and a Kromasil C-18 separation column. The protein digestibility was measured by sequential exposure to pepsin-trypsin proteinase system under simulated gastric digestion.
 Long-time dry aging triggered proteolysis under the action of endogenous enzymes. The electropherogram analysis showed that the proteolytic changes in high-molecular myofibrillar proteins of high-quality beef became more pronounced after a longer maturation period. The distribution of protein fractions by dry aging stages indicated a different rate of degradation of contractile, regulatory, and cytoskeletal proteins. As a result, the structural integrity of muscle fibers degraded, the meat grew tender, and the proteins became more available to digestive enzymes. The amino acid and protein digestibility analyses in vitro demonstrated an increase in the nutritional value of beef and the availability of proteins to the action of proteinases after 42 days of dry aging.
 Long-term dry aging of high-quality beef increased the digestibility of muscle proteins as a result of proteolysis that accompanied the accumulation of low-molecular fractions. According to the amino acid analysis, the optimal result was most pronounced on day 42 as proven by the moderate oxidative changes in proteins.

Calpain-dependent degradation of cytoskeletal proteins as a key mechanism for a reduction in intrinsic passive stiffness of unloaded rat postural muscle.

In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.

Oxidative Stress Contributes to Cytoskeletal Protein Degradation of Esox lucius through Activation of Mitochondrial Apoptosis during Postmortem Storage.

This study investigated the role of oxidative stress in the mitochondrial apoptotic pathways and structural protein degradation of fish during postmortem storage by measuring oxidative stress levels, mitochondrial antioxidant enzyme activity, mitochondrial dysfunction, apoptotic factors, and structural protein degradation (n = 3). The results revealed that reactive oxygen species (ROS) increased gradually within the first 12 h and then decreased (p < 0.05) in mitochondria. Lipid peroxidation was increased, and superoxide dismutase, catalase, and glutathione peroxidase activities were decreased in mitochondria (p < 0.05). Furthermore, oxidative stress induced mitochondrial membrane opening, mitochondrial swelling, as well as the depolarization of mitochondrial potential. This led to an increase in the release of cytochrome c from mitochondria and caspase-3 activation. Ultimately, oxidative stress promoted small protein degradation (troponin-T and desmin) and induced myofibril susceptibility to proteolysis. These observations confirmed that oxidative stress mediated the activation of mitochondrial apoptotic factors-promoted protein degradation, initiating the deterioration of fish muscle through the mitochondrial apoptotic pathway.

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α‐actinin‐4 and talin‐1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin‐1 is a specific proteolytic target of calpain‐1, we investigated a potential trigger of calpain activation and talin‐1 degradation using human ex vivo precision‐cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain‐1 by transforming growth factor‐β generated proteolytic fragments of talin‐1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain‐1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

Hirudin attenuates puromycin aminonucleoside-induced glomerular podocyte injury by inhibiting MAPK-mediated endoplasmic reticulum stress.

Damage to podocytes is an important determinant of renal pathology. The puromycin aminonucleoside (PAN) mice nephropathy model is commonly used in the study of renal disease with podocyte injury. Hirudin has a broad nephroprotective effect and has been shown to treat renal interstitial fibrosis in previous studies. Mice were given PAN by gavage to prepare animal models, and MPC5 cells were incubated with PAN in vitro. Twenty-four hours urine was collected for analysis of urinary protein levels. Renal pathological changes were observed by hematoxylin and eosin staining. Immunofluorescence detection of nephrin in kidney tissues and cells. Apoptosis was analyzed with over TUNEL. Cytoskeleton, endoplasmic reticulum stress (ERS), p38 MAPK signaling, and apoptosis-related proteins were assessed by western blot analysis. The data suggested that hirudin attenuated reduced renal injury and increased urine protein in PAN mice. Hirudin also attenuated cytoskeletal protein (synaptopodin, nephrin, and podocin) disruption, ERS activation, and apoptosis in PAN mice and PAN-induced podocytes. In addition, hirudin inhibited the expression of p38 MAPK signaling key proteins upregulated by PAN, thereby suppressing ERS. The p38 MAPK agonist was able to partially antagonize the inhibition of p38 MAPK signaling by hirudin in PAN-induced podocytes, thereby reactivating the ERS inhibited by hirudin, promoting cytoskeletal protein degradation and increasing the level of apoptosis. In conclusion, hirudin could decrease podocyte injury by inhibiting p38 MAPK signaling-mediated ERS, resulting in the protection of the kidney from PAN damage. These findings may provide an experimental basis for hirudin treatment of podocyte injury diseases.

Receptor tyrosine kinase ROR1 ameliorates A\u03b21\u201342 induced cytoskeletal instability and is regulated by the miR146a-NEAT1 nexus in Alzheimer\u2019s disease

Alzheimer’s disease (AD) involves severe cytoskeletal degradation and microtubule disruption. Here, we studied the altered dynamics of ROR1, a Receptor Tyrosine Kinase (RTK), and how it could counter these abnormalities. We found that in an Aβ1–42 treated cell model of AD, ROR1 was significantly decreased. Over expressed ROR1 led to the abrogation of cytoskeletal protein degradation, even in the presence of Aβ1–42, preserved the actin network, altered actin dynamics and promoted neuritogenesis. Bioinformatically predicted miRNAs hsa-miR-146a and 34a were strongly up regulated in the cell model and their over expression repressed ROR1. LncRNA NEAT1, an interactor of these miRNAs, was elevated in mice AD brain and cell model concordantly. RNA Immunoprecipitation confirmed a physical interaction between the miRNAs and NEAT1. Intuitively, a transient knock down of NEAT1 increased their levels. To our knowledge, this is the first instance which implicates ROR1 in AD and proposes its role in preserving the cytoskeleton. The signalling modalities are uniquely analyzed from the regulatory perspectives with miR-146a and miR-34a repressing ROR1 and in turn getting regulated by NEAT1.

Impact of postmortem degradation of cytoskeletal proteins on intracellular gap, drip channel and water-holding capacity

The present study aimed to investigate the impact of the degradation of cytoskeletal proteins (desmin, integrin, vinculin, and talin) on the formation of intracellular gap and drip channel and water-holding capacity in pork. The intensity of intact cytoskeletal proteins and the width of intracellular gap and drip channel were measured in high drip loss and low drip loss groups. The data indicate that the width of intracellular gap and drip channel explained 17% and 62% variation in drip loss, respectively, while the intensity of intact desmin, integrin, and vinculin explained 47%, 34%, and 47% variation in drip loss, respectively. The postmortem formation of intracellular gap is mainly affected by the changes in integrin and vinculin, while the formation of the drip channel is influenced by the changes in desmin. These findings suggest that postmortem formation of wide intracellular gap and drip channels is linked to increased drip loss.

Molecular Mechanisms of Muscle Tone Impairment under Conditions of Real and Simulated Space Flight.

Kozlovskaya et al. [1] and Grigoriev et al. [2] showed that enormous loss of muscle stiffness (atonia) develops in humans under true (space flight) and simulated microgravity conditions as early as after the first days of exposure. This phenomenon is attributed to the inactivation of slow motor units and called reflectory atonia. However, a lot of evidence indicating that even isolated muscle or a single fiber possesses substantial stiffness was published at the end of the 20th century. This intrinsic stiffness is determined by the active component, i.e. the ability to form actin-myosin cross-bridges during muscle stretch and contraction, as well as by cytoskeletal and extracellular matrix proteins, capable of resisting muscle stretch. The main facts on intrinsic muscle stiffness under conditions of gravitational unloading are considered in this review. The data obtained in studies of humans under dry immersion and rodent hindlimb suspension is analyzed. The results and hypotheses regarding reduced probability of cross-bridge formation in an atrophying muscle due to increased interfilament spacing are described. The evidence of cytoskeletal protein (titin, nebulin, etc.) degradation during gravitational unloading is also discussed. The possible mechanisms underlying structural changes in skeletal muscle collagen and its role in reducing intrinsic muscle stiffness are presented. The molecular mechanisms of changes in intrinsic stiffness during space flight and simulated microgravity are reviewed.

The Effect of µ/m-Calpain on Protein Degradation of Chicken Breast Meat.

This study was designed to determine the effects ofµ/m-calpain on the degradation of cytoskeletal proteins in pectoralis major. Four chickens were slaughtered and the breasts were removed and stored for 12hr at 4 °C. Each sample was divided into three groups and respectively immersed in control reagent, calpain inhibitor, and caspase inhibitor at 4°C. The samples were used to evaluate troponin-T and desmin degradation, calpain activity, and myofibril ultrastructure at 12hr, day 1, day 3, and day 7. Casein zymography revealed thatµ-calpain could not be detected in all samples after 12hr postmortem. The calpain inhibitor inhibitedµ/m-calpain activity and reduced troponin-T and desmin degradation during 7day postmortem. The caspase inhibitor inhibitedµ/m-calpain activity and, troponin-T and desmin degradation before day 3 postmortem. The findings suggest that,µ/m-calpain had an effect on cytoskeletal protein degradation after 12hr postmortem.

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis.

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.

Apoptosis and Acute Brain Ischemia in Ischemic Stroke.

Apoptosis may contribute to a significant proportion of neuron death following acute brain ischemia (ABI), but the underlying mechanisms are still not fully understood. Brain ischemia may lead to stroke, which is one of the main causes of long-term morbidity and mortality in both developed and developing countries. Therefore, stroke prevention and treatment is clinically important. There are two important separate areas of the brain during ABI: the ischemic core and the ischemic penumbra. The ischemic core of the brain experiences a sudden reduction of blood flow, just minutes after ischemic attack with irreversible injury and subsequent cell death. On the other hand, apoptosis within the ischemic penumbra may occur after several hours or days, while necrosis starts in the first hours after the onset of ABI in the ischemic core. ABI is characterized by key molecular events that initiate apoptosis in many cells, such as overproduction of free radicals, Ca2+ overload and excitotoxicity. These changes in cellular homeostasis may trigger either necrosis or apoptosis, which often depends on cell type, cell age, and location in the brain. Apoptosis results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells. This review focuses on recent findings based on animal and human studies regarding the apoptotic mechanisms of neuronal death following ABI and the development of potential neuroprotective agents that reduce morbidity. The effects of statins on stroke prevention and treatment as well as on apoptotic mediators are also considered.

Influence of Postmortem Aging of Fresh Pork Loin on Instrumental Tenderness and Abundance of a Soluble Desmin Degradation Product

ObjectivesIt is well understood that aging fresh pork loins will improve tenderness. The explanation for this phenomenon is degradation of myofibrillar, cytoskeletal, and intermediate filament proteins by endogenous proteolytic enzymes. Recently, the abundance of a desmin fragment in the sarcoplasmic fraction of aged pork has been linked to differences in pork tenderness. The objective of this experiment was to document the abundance of this desmin degradation product in the sarcoplasmic fraction during aging of fresh pork loin and determine its relationship to fresh pork tenderness.Materials and MethodsLoins (n = 20) were collected 1 d postmortem at a commercial processing facility. Criteria for inclusion in the study was an average pH between 5.70 and 5.85 and a visual color score (National Pork Board) between 3 and 4. Two loin chops containing only the longissimus muscle (2.54 cm and adjacent 1 cm chop) from each loin were aged 1, 3, 7, or 14 d. Upon completion of aging, 2.54 cm chops (never frozen) were used to determine Hunter L, a, b, pH, color scores, and marbling scores. These chops were then cooked to 68°C and evaluated for cook loss and star probe (kg). The 1 cm thick chops were frozen and homogenized in liquid nitrogen at the end of each aging period. Proteins were fractionated to isolate proteins soluble in a low ionic strength buffer (40 mM Tris, 1 mM EDTA, pH 8.0). Abundance of a desmin degradation product (34 kDa) in the sarcoplasmic fraction was determined by immunoblotting and normalized to the abundance of a reference sample on each gel. Data were analyzed with a fixed effect of days of aging, and a random effect of loin. Pearson correlations for the quality variables were calculated.ResultsStar probe values declined with aging (7.9 kg, 6.4 kg, 5.6 kg, and 5.1 kg after aging 1, 3, 7, and 14 d respectively). Each aging period showed a significant decline in star probe (P < 0.05). The abundance of the desmin degradation product in the sarcoplasmic fraction significantly increased between 1, 3, and 7 d aging (P < 0.01). No difference in desmin fragment abundance was observed in a comparison of samples aged 7 and 14 d. Across all days of aging, star probe was positively correlated with cook loss (r = 0.59), and weakly correlated with pH measured on the day of aging (r = 0.29). Across all aging periods, desmin degradation product abundance was significantly negatively correlated (r = –0.49) with star probe values. Abundance of the desmin degradation product in the sarcoplasmic fraction measured after aging 1 d postmortem was significantly negatively correlated with star probe measured 3,7, and 14 d postmortem (r = –0.46, –0.44, and –0.45 respectively). Presence of soluble desmin in early postmortem pork may aid in predicting pork loin tenderness after aging.ConclusionTherefore, results of this study demonstrate promise of using the abundance of a desmin degradation product in the sarcoplasmic fraction of early postmortem pork to predict fresh pork tenderness.

Effect of fast pH decline during the early postmortem period on calpain activity and cytoskeletal protein degradation of broiler M. pectoralis major

The objective of this study was to determine the effects of fast pH decline during the early postmortem period on calpain activity and the degradation of cytoskeletal proteins in broilers. Eighty broilers were randomly categorized into two groups: physical restraint (PR) and free struggle (FS). M. pectoralis major (PM) was used for determination of calpain activity, shear value, ultrastructure of myofibrils, and the degradation of desmin, titin, nebulin, and troponin-T. The pH (6.05) of FS group is significantly low than PR group (6.38) at 0.3 h postmortem. Fast pH decline during the early postmortem period led to a decrease of μ/m-calpain activities at 0.3 and 3 h postmortem (P < 0.05), but did not affect the ultimate μ/m-calpain activity. An initial fast decrease in pH increased the degradation of desmin, titin, nebulin, and increased the 30 kDa degradation fragments of troponin-T. Therefore, the fast pH decline during the early postmortem period decreased the μ/m-calpain activity and increased the degradation of cytoskeletal proteins in broiler muscle. It is possible that the fast pH decline experienced an earlier activation of calpains that resulted in earlier protein degradation and ultimately lower shear force.

Proteomic Analysis of Protein Expression Throughout Disease Progression in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. Mice in the transgenic AβPPswe/PS1dE9 mouse line express a chimeric mouse/human amyloid-β protein precursor (Mo/HuAβPP695swe) and mutant human presenilin 1 (PS1-dE9) associated with early-onset AD. Knowing the protein expression in these mice may offer better understanding of the pathological changes in AD. In this study, we used two-dimensional gel electrophoresis combined with mass spectrometry techniques to compare protein expression in AβPPswe/PS1dE9 mice with age-matched wild-type mice throughout the disease progression. We identified 15 proteins that were significantly different between the AβPPswe/PS1dE9 mice and age-matched controls and also changed with disease development. Among those, the expression levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times higher than those in normal mice: DCC-interacting protein 13-beta, serum albumin, creatine kinase B-type, heat shock 70 kDa protein 1A, T-complex protein 1 subunit beta, adenylate kinase isoenzyme 1, pyruvate dehydrogenase E1 component subunit beta mitochondrial, and V-type proton ATPase catalytic subunit A. Levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times lower than those in normal mice: dihydropyrimidinase-related protein 2, actin cytoplasmic 2, isoform 1 of V-type proton ATPase catalytic subunit, tubulin alpha-1C chain, F-actin-capping protein subunit alpha-2, ubiquitin carboxyl-terminal hydrolase isozyme L1, and actin cytoplasmic 1. These proteins are involved in regulating various cellular functions, including cytoskeletal structure, energy metabolism, synaptic components, and protein degradation. These findings indicate altered protein expression in the pathogenesis of AD and illuminate novel therapeutic avenues for treatment in AD.

Osnove apoptoze

Apoptosis is evolutionary conserved, programmed pattern of cell death with an essential role in various physiological processes, such as normal cell turnover and embryonic development, hormone-regulated cell demise, aging, immune system functioning and development and removal of defective and harmful cells. There are two general pathways for activation of apoptosis: the intrinsic and extrinsic pathways. While the intrinsic apoptotic pathway can be triggered by a cytotoxic accumulation of intracellular Ca 2+ , followed permeabilization of mitochondrial membrane and release of pro-apoptotic proteins into the cytosol from mitochondria, the extrinsic mechanisms of apoptosis include the participation of death receptors of tumor necrosis factor-a (TNF-a), receptor superfamily such as TNFR-1, Fas, and TNF-related apoptosis-inducing ligand receptors (TRAIL-R) located on the plasma membrane. There is also the perforin-granzyme pathway that involves T-cell mediated cytotoxicity. All three pathways converge on the same execution pathway, resulting in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells. In this review we summarize data from recent studies focusing on apoptotic proteins that have been identified and molecular mechanisms of apoptosis. Understanding apoptotic mechanism might provide useful information and a new approach to prevention and development of new therapies for variety of diseases.

Proteomic pattern changes associated with obesity‐induced asthenozoospermia

Obesity, an increasingly frequent societal disease can also be accompanied by declines in spermatozoa quality and male subfecundity. To determine if there are obesity-associated proteomic changes potentially affecting sperm quality and motility, differential proteomic analysis was performed on spermatozoa from both obesity-associated asthenozoospermia and clinically healthy individuals, using a label-free quantitative LC-MS/MS approach. We resolved 1975 proteins in the human sperm proteome, amongst which, 105 proteins were less abundant, whereas 22 other proteins increased in obesity-associated asthenozoospermia. Functional category analyses indicated that the differentially expressed proteins are mainly related to cytoskeletal regulation, vesicle biogenesis, metabolism, and protein degradation involved in spermiogenesis and sperm motility. Furthermore, declines in endoplasmic reticulum protein 57 (ERp57) and actin-binding-related protein T2 (ACTRT2) expression were verified by immunofluorescence, Western blot, and flow cytometry analyses. It is evident that ERp57 is localized in the acrosome region, neck and principal piece of human spermatozoa, whereas ACTRT2 is localized in the post-acrosomal region and middle piece. Thus, these differences in protein expression in asthenozoospermia may contribute to the underlying sperm quality defects afflicting these individuals. Notably, declines in ERp57 and ACTRT2 expression in obesity-associated asthenozoospermia may play critical roles in reducing sperm motility.

Degradation of βII-Spectrin Protein by Calpain-2 and Caspase-3 Under Neurotoxic and Traumatic Brain Injury Conditions.

A major consequence of traumatic brain injury (TBI) is the rapid proteolytic degradation of structural cytoskeletal proteins. This process is largely reflected by the interruption of axonal transport as a result of extensive axonal injury leading to neuronal cell injury. Previous work from our group has described the extensive degradation of the axonally enriched cytoskeletal αII-spectrin protein which results in molecular signature breakdown products (BDPs) indicative of injury mechanisms and to specific protease activation both in vitro and in vivo. In the current study, we investigated the integrity of βII-spectrin protein and its proteolytic profile both in primary rat cerebrocortical cell culture under apoptotic, necrotic, and excitotoxic challenge and extended to in vivo rat model of experimental TBI (controlled cortical impact model). Interestingly, our results revealed that the intact 260-kDa βII-spectrin is degraded into major fragments (βII-spectrin breakdown products (βsBDPs)) of 110, 108, 85, and 80 kDa in rat brain (hippocampus and cortex) 48 h post-injury. These βsBDP profiles were further characterized and compared to an in vitro βII-spectrin fragmentation pattern of naive rat cortex lysate digested by calpain-2 and caspase-3. Results revealed that βII-spectrin was degraded into major fragments of 110/85 kDa by calpain-2 activation and 108/80 kDa by caspase-3 activation. These data strongly support the hypothesis that in vivo activation of multiple protease system induces structural protein proteolysis involving βII-spectrin proteolysis via a specific calpain and/or caspase-mediated pathway resulting in a signature, protease-specific βsBDPs that are dependent upon the type of neural injury mechanism. This work extends on previous published work that discusses the interplay spectrin family (αII-spectrin and βII-spectrin) and their susceptibility to protease proteolysis and their implication to neuronal cell death mechanisms.

New Polymorphisms in Regulatory Region of CAPN3 Gene with no Effect on Gene Expression in Breast Muscle of Broiler Chickens

Abstract Calpains are enzymes that belong to calcium-dependent, non-lysosomal cysteine proteases. The CAPN3 gene encodes a major intracellular specific muscle protease with a high capacity for degradation of cytoskeletal and muscle fibre proteins. Therefore, they play an important role in fusion of myoblasts, proliferation, cell growth and migration. In chickens, the gene encoding for calpain 3 is localized on chromosome 5. Due to the function of encoded protein, the CAPN3 has beenchosen as a candidate gene for meat quality in chickens. Consequently, the aim of our study was to identify new polymorphisms in the regulatory region of CAPN3 gene and to investigate their impact on CAPN3 transcript abundance in breast muscles. The experiment used broilers of two genetic lines: fast- and slow-growing. The polymorphisms were identified by screening with High Resolution Melting (HRM) and sequencing based on the Sanger method. The CAPN3 gene expression was conducted by using succinate dehydrogenase complex, subunit A (SDHA) and 60S ribosomal protein L4 (RPL4) genes as endogenous controls. Four new polymorphisms were found: g.322176G&gt;A in promoter region (GenBank: AADN03004661.1), c.176*C&gt;T, c.144*G&gt;C and c.137*_147*delCAGCCCTGCTT in 3`UTR sequence. The new polymorphisms were identified by using restriction enzymes ScrFI, BslI, AcuI, HpyAV, respectively. The frequency of polymorphisms found in 3`UTR region was similar in both lines. According to polymorphisms identified in 3`UTR region the alleles with deletion, C and C (c.137*_147*del, c.144*G&gt;C and c.176*C&gt;T) were rare. The polymorphism identified in the promoter region and 3`UTR regions changed a few binding sites for transcription factors, but did not alter any binding sites for the other important translation regulators such as miRNA. Analysis of the effect of new polymorphisms on CAPN3 gene expression showed that in fast-growing line the chickens with GG genotype according to polymorphism g.322176G&gt;A inthe promoter region, were characterized by the highest CAPN3 transcript abundance. Other polymorphisms in 3&gt;UTR region seem to have no effect on CAPN3 gene expression.