Protein Turnover Research Articles

Greater energy surplus promotes body protein accretion in healthy young men: A randomized clinical trial

Background & aimsCaloric overfeeding combined with adequate protein intake increases not only body fat mass but also fat-free mass. However, it remains unclear whether the increase in fat-free mass due to overfeeding is associated with an increase in total body protein mass. We evaluated the hypothesis that overfeeding would promote an increase in total body protein mass. MethodsIn our randomized controlled trial, 23 healthy young men were fed a diet equivalent to their energy requirements with a +10 % energy surplus from protein alone or a +40 % energy surplus (+10 % from protein, +30 % from carbohydrate) for 6 weeks. We estimated total body protein mass by a four-compartment model using dual-energy X-ray absorptiometry, deuterium dilution, and hydrostatic underwater weighing. ResultsThe 40 % energy surplus over 6 weeks significantly increased body protein mass compared to baseline by 3.7 % (0.44 kg; 95 % confidence interval [CI], 0.21–0.67 kg; P = 0.003); however, the 10 % energy surplus did not result in a significant change (0.00 kg; 95 % CI, −0.38–0.39 kg; P = 0.980). A significant interaction between intervention duration (time) and energy surplus (group) was observed for total body protein mass (P = 0.035, linear mixed-effects model), with a trend toward a significant difference in total body protein mass gain between groups (P = 0.059, Wilcoxon rank sum test). The increase in body protein mass due to the energy surplus was correlated with an increase in fat mass (r = 0.820, p = 0.002). ConclusionsA higher energy intake was found to promote an increase in body protein mass in healthy men consuming excess protein, suggesting the importance of energy surplus in body protein accumulation. This effect of energy surplus may be related to factors such as increased body fat mass and the associated secretion of adipokines. Trial registrationThe trial was registered with the University Hospital Medical Information Network Clinical Trial Registry as UMIN000034158.

Transcriptomic and protein analysis of Trametes versicolor interacting with a Hypholoma fasciculare mycelium foraging in soil

The decomposition of large woody material is an important process in forest carbon cycling and nutrient release. Cord-forming saprotrophic basidiomycete fungi create non-resource limited mycelial networks between decomposing branches, logs and tree stumps on the forest floor where colonisation of new resources is often associated with the replacement of incumbent decay communities. To date, antagonism experiments have mostly placed competing fungi in direct contact, while in nature cord-forming saprobes encounter colonised wood as mycelia in a network. Transcriptomic and peptide analyses were conducted on soil-based microcosms were foraging cord-forming Hypholoma fasciculare encountered a wood block colonised by Trametes versicolor. Protein turnover featured strongly for both species and genes putatively involved in secondary metabolite production were identified. H. fasciculare demonstrated an exploitative profile with increased transcription of genes associated with carbohydrate metabolism and RNA and ribosome processing. T. versicolor showed a shift in signalling, energy generation and amino acid metabolism. By identifying genes and proteins putatively involved in this fungal interaction, this work may help guide the discovery of bioactive molecules and mechanisms underpinning community succession.

AAMP and MTSS1 Are Novel Negative Regulators of Endothelial Barrier Function Identified in a Proteomics Screen.

Cell-cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability.

Skeletal Muscle Memory: An Update From the Antidoping Perspective.

This narrative review explores the concept of muscle memory, focusing on the physiological and biochemical mechanisms underlying information retention in skeletal muscle tissue as it relates to antidoping. The discussion encompasses the role of satellite cells (SCs) in myonuclei recruitment, resulting in increased myonuclear density and heightened muscle protein turnover. The myonuclear domain theory suggests that myonuclei acquired during hypertrophy may persist, contributing to enhanced muscle protein synthesis (MPS) and potential benefits of muscle memory. The impact of sustained training, protein intake, and resistance exercise on muscle memory, especially in elite athletes, is considered. The review also delves into the influence of anabolic androgenic steroids (AAS) on muscle tissue, highlighting their role in elevating the performance threshold and supporting recovery during intense training through increased muscle protein turnover rates. Additionally, genetic and epigenetic modifications, such as DNA methylation, are explored as potential contributors to muscle memory. The complex interplay of continuous training, AAS use, and genetic factors offers avenues for further research, especially in the context of antidoping efforts. The understanding of muscle memory has implications for maintaining performance gains and addressing ethical challenges in sports.

Effects of intermittent exposure to hypobaric hypoxia and cold on skeletal muscle regeneration: Mitochondrial dynamics, protein oxidation and turnover

Muscle injuries and the subsequent regeneration events compromise muscle homeostasis at morphological, functional and molecular levels. Among the molecular alterations, those derived from the mitochondrial function are especially relevant. We analysed the mitochondrial dynamics, the redox balance, the protein oxidation and the main protein repairing mechanisms after 9 days of injury in the rat gastrocnemius muscle. During the recovery rats were exposed to intermittent cold exposure (ICE), intermittent hypobaric hypoxia (IHH), and both simultaneous combined stimuli. Non-injured contralateral legs were also analysed to evaluate the specific effects of the three environmental exposures. Our results showed that ICE enhanced mitochondrial adaptation by improving the electron transport chain efficiency during muscle recovery, decreased the expression of regulatory subunit of proteasome and accumulated oxidized proteins. Exposure to IHH did not show mitochondrial compensation or increased protein turnover mechanisms; however, no accumulation of oxidized proteins was observed. Both ICE and IHH, when applied separately, elicited an increased expression of eNOS, which could have played an important role in accelerating muscle recovery. The combined effect of ICE and IHH led to a complex response that could potentially impede optimal mitochondrial function and enhanced the accumulation of protein oxidation. These findings underscore the nuanced role of environmental stressors in the muscle healing process and their implications for optimizing recovery strategies.

The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic vascular dementia characterized by age-related degeneration of vascular mural cells and accumulation of a NOTCH3 mutant protein. NOTCH3 functions as a signaling receptor, activating downstream gene expression in response to ligands like JAG1 and DLL4, which regulate the development and survival of mural cells. This signal transduction process is thought to be connected with NOTCH3 endocytic degradation. However, the specific cellular circumstances that modulate turnover and signaling efficacy of NOTCH3 mutant protein remain largely unknown. Here, we found elevated NOTCH3 and Radical fringe (RFNG) expression in senescent human pericyte cells. We then investigated impacts of RFNG on glycosylation, degradation, and signal activity of three NOTCH3 CADASIL mutants (R90C, R141C, and C185R) in EGF-like repeat-2, 3, and 4, respectively. LC–MS/MS analysis showed that RFNG modified NOTCH3 WT and C185R to different degrees. Additionally, coculture experiments demonstrated that RFNG significantly promoted JAG1-dependent degradation of NOTCH3 WT but not that of R141C and C185R mutants. Furthermore, RFNG exhibited a greater inhibitory effect on JAG1-mediated activity of NOTCH3 R141C and C185R compared to that of NOTCH3 WT and R90C. In summary, our findings suggest that NOTCH3 R141C and C185R mutant proteins are relatively susceptible to accumulation and signaling impairment under cellular conditions of RFNG and JAG1 coexistence.

Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection.

ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by poly(ADP-ribose) polymerases (PARPs) that modulates protein function, abundance, localization and turnover. Here we show that influenza A virus infection causes a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry defined for the first time the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4,300 modification sites on ~1,080 host proteins, as well as over 100 modification sites on viral proteins. Our data indicate that the global increase likely reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrated that modification of the viral replication machinery antagonizes its activity and further revealed that the anti-viral activity of PARPs and ADP-ribosylation is counteracted by the influenza A virus protein NS1, assigning a new activity to the primary viral antagonist of innate immunity. We identified PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicated faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1.

Overexpression of SIMK in menadione-treated alfalfa enhances antioxidant machinery and leads to oxidative stress resistance

Mitogen-activated protein kinases (MAPKs) transduce stress and developmental signals related to the production of reactive oxygen species (ROS). Alfalfa (Medicago sativa L.) is a valuable forage and human nutrition crop, however, the involvement of MAPKs in plant resistance to oxidative stress is poorly understood in this species. Therefore, we elucidated the role of STRESS-INDUCED MAPK (SIMK) in alfalfa response to menadione, a compound inducing ROS generation, exploiting transgenic alfalfa lines with contrasting SIMK abundance. SIMK was activated by short-term menadione treatment and relocated from the nucleus to the cytoplasm. Proteomic analysis revealed that menadione caused changes in the abundance of proteins involved in metabolism, oxidative stress, biotic stress response, detoxification of carbonyl species, glutathione homeostasis, chloroplast protein turnover, photosynthesis, and membrane trafficking. Genetic manipulations of SIMK altered the abundance of proteins involved in mitochondrial and chloroplast protein import and processing, as well as GLUTATHIONE S-TRANSFERASES (GSTs). Increased GST abundance and activity in roots, and modifications in mitochondrial and chloroplast protein turnover might be responsible for the elevated oxidative stress resistance of alfalfa line overexpressing SIMK. This was supported by the reduced ROS levels in this line. These results reveal a complex nature of plant stress response and suggest a new role of SIMK in the alfalfa resistance to menadione-induced oxidative stress.

Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts.

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this "cataract-reversing" phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress-related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.

Autophagy in Oligodendrocyte Lineage Cells Controls Oligodendrocyte Numbers and Myelin Integrity in an Age-dependent Manner.

Oligodendrocyte lineage cells, including oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs), are essential in establishing and maintaining brain circuits. Autophagy is a conserved process that keeps the quality of organelles and proteostasis. The role of autophagy in oligodendrocyte lineage cells remains unclear. The present study shows that autophagy is required to maintain the number of OPCs/OLs and myelin integrity during brain aging. Inactivation of autophagy in oligodendrocyte lineage cells increases the number of OPCs/OLs in the developing brain while exaggerating the loss of OPCs/OLs with brain aging. Inactivation of autophagy in oligodendrocyte lineage cells impairs the turnover of myelin basic protein (MBP). It causes MBP to accumulate in the cytoplasm as multimeric aggregates and fails to be incorporated into integral myelin, which is associated with attenuated endocytic recycling. Inactivation of autophagy in oligodendrocyte lineage cells impairs myelin integrity and causes demyelination. Thus, this study shows autophagy is required to maintain myelin quality during aging by controlling the turnover of myelin components.

Abstract A060: Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), with a subset contributing significantly to tumor progression. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is associated with increased tumor aggressiveness. Our group previously demonstrated PTEN loss in SMA+ positive CAFS correlated with worsened outcomes in patients and in mouse PDAC models. Our group previously demonstrated the loss of PTEN in CAFS led to activation of Stat3, resulting in an immunosuppressive microenvironment (Lefler et al, 2022 PMID: 35803738). However, little is known of the signals produced by tumor cells or other cell types in the TME that trigger loss of PTEN in CAFs. This study aims to to elucidate the molecular mechanisms underlying PTEN loss in CAFs in PDAC. To address this gap, we employed single cell RNA sequencing of tumors from a mouse PDAC model and identified a (PDRGFRa+Lys6+Stat3+Pten-low) CAF subpopulation. Comparing normal pancreas epithelial cells to tumor cells in this data set, we identified the IL6 family of cytokines, (IL6; Oncostatin-M, OSM; and Leukemia Inhibitory Factor, LIF) and Transforming Growth Factor beta 1 (TGFb1) as potential ligands that regulated PTEN, while myeloid cells were a source of the IL6 family member Oncostatin M (OSM). Treatment of isolated CAFs with these factors revealed no significant difference in PTEN mRNA expression. Thus, we developed an in vitro reporter system in which a PTEN-GFP fusion protein is expressed in CAFs. Treating these CAFs with IL6, OSM and TGFb1 (but not LIF) led to a significant decrease in GFP fluorescence. Analysis of endogenous PTEN by western blot confirmed these results. We posited that expression of PTEN E3 ligases might be increased by treatment with these factors, leading to destruction of PTEN. In support of this hypothesis, the expression of E3 ligases WWP1 and WWP2 was increased by treatment with IL6 and OSM. Additionally, preliminary data has revealed that PTEN is oxidized in isolated CAFs, which causes loss of PTEN activity and potentially increased protein turnover. Current experiments are aimed at testing these potential mechanisms. To conclude, PTEN is dysregulated post-transcriptionally in CAFs. The mechanism of PTEN dysregulation could be via increased expression of E3 ligases that target PTEN and/or PTEN oxidation. Future efforts will be aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models. These pathways and interactions could be further exploited for therapeutic benefit in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A060.

PSLBI-22 Identifying plasma metabolites influencing body weight in Salmonella challenged growing pigs: A machine learning approach

Abstract Body weight (BW) is an important component of pig productivity. Understanding the metabolic factors affecting growth rate is key to guiding nutritional strategies and bringing economic benefits to pig farms. Machine learning (ML) algorithms have been used to capture patterns in large datasets and predict animal production traits based on integrated information of performance data and blood records. However, utilizing ML models and metabolomic profiling to predict BW of pigs in small datasets with challenged animals has yet to be reported. Therefore, this study investigated use of plasma metabolites in predicting BW of pigs at the growing stage. This simulation used data from gilts (n = 40; BW = 25.6 ± 2.3 kg) raised over a 28-d trial. Plasma samples and BW were collected on d 0, 14, and 28 after 8 h of fasting. Ninety plasma metabolites were identified and quantified from an untargeted metabolomics assay. The development dataset included 30, and the validation dataset contained 10 unique animals with repeated measures in time. The metabolites were log-transformed, Pareto-scaled and missing values were imputed using the minimum nonzero value method. The BW prediction model was performed in R with the caret package. The optimal hyper-parameter configuration for the random forest and neural network algorithms was evaluated based on repeated cross-validation. The final model was the random forest composed of 400 trees with 45 randomly selected predictors. Model accuracy was estimated on repeated cross-validation with 10-folds and 5 repeats and compared with the validation set based on the coefficient of determination (R2), the root mean squared error (RMSE), and Pearson correlation (r). The metabolites with greater contribution with variable importance score above 10 to the model development were extracted. Aiming to understand differences in BW categories, the model response was visualized with an alluvial diagram for the 3 variables with the greatest metabolite importance, and BW was classified as high, medium, and low. The main metabolites driving BW were lysine, oleic acid, aminomalonic acid, myo-inositol, and linoleic acid. The RMSE after cross-validation was 7.22 kg of BW (r = 0.70), and R2 = 0.39. For pigs with a high BW, the predicted BW was explained by generally less plasmatic concentrations of lysine, oleic acid, and aminomalonic acid. Lysine is the main amino acid for muscle protein accretion, whereas oleic acid is for energy substrate. Aminomalonic acid is a metabolite resulting from glycine metabolism and is important in reversing oxidative damage caused by pathogens. Therefore, decreased plasma lysine, amino malonic acid, and oleic acid may indicate faster growth and lean deposition, leading to heavier BW in pigs. Using random forests helped to connect the plasma dynamics to BW and is a promising avenue to understand the main drivers in BW of group-housed pigs.

352 Supplementing bovine satellite cells with anabolic hormones and/or zinc at differentiation impacts abundance of mRNA involved in growth, mineral transport, and oxidative stress

Abstract Zinc (Zn) is the most utilized metal in biological processes and supports growth through critical roles in protein turnover. The Zn requirements of skeletal muscle may be augmented when growth promoting technologies, such as anabolic hormones are being utilized. Both anabolic hormones and Zn affect protein synthesis and degradation in skeletal muscle. However, the molecular mechanisms through which Zn may interact with anabolic hormones to support skeletal muscle growth remains unknown. The objective of this study was to determine whether trenbolone acetate (TBA), estradiol (E2) and Zn interact to promote differentiation of bovine satellite cell (BSC) cultures by assessing abundance of mRNA involved in growth, mineral transport, differentiation, protein turnover and oxidative stress. To do this, primary BSC cultures were isolated from three different steers and grown to 80% confluency and induced to differentiate in 3% horse serum, at which time a factorial design was employed to assess the impact of Zn (0, 10, 20 or 40 μM) as ZnCl2 and/or the impact of hormone (10 nM TBA,10 nM E2, 10 nM TBA and 10 nM E2). Zinc concentrations represent physiologically relevant plasma Zn concentrations. Total mRNA was isolated from the treated cultures at 4 h, 12 h and 24 h post-treatment. Assays utilizing cultures from each steer were run in duplicate (n = 6). The mRNA abundance of 45 genes were examined and analyzed using the PROC MIXED procedure of SAS with time as a repeated measure to evaluate the fixed effects of hormone, mineral, time, and all interactions. No effects (P ≥ 0.19) of hormone x mineral x time, hormone x time, hormone x mineral or hormone were noted. A mineral x time effect (P ≤ 0.04) was noted for the igf1r, slc39a7, mapk1, mmp2, and G6pd. A mineral effect was found for hormone receptors (ir, ar, esr1, esr2, and gper; P ≤ 0.03), mRNA involved in mineral transport (slc30a10, slc30a7, sod1, sod2, slc2a4, and slc39a8; P ≤ 0.009), and genes related to skeletal muscle differentiation (pax7, myf5, myf6, spry1, myog, mef2a, and myod; P ≤ 0.02) and protein turnover (akt1, fox01, fox04, mstn,fbox32, fox03, mtor, trim63, and CKM; P ≤ 0.03). These differences are generally driven by abundance being increased (P ≤ 0.05) in cultures treated with 40 or 20 μM Zn compared with cultures receiving 0 or 10 μM Zn. These results indicate that while Zn and hormone do not appear to interact to influence gene expression of differentiating primary BSC cultures at the time points measured, increased concentrations of Zn appear to increase mRNA abundance of hormone receptors, mineral transporters, and genes involved in skeletal muscle differentiation and protein turnover.

54 Development of an equine metabolism model to describe post-absorptive nutrient dynamics

Abstract Nutrition modelling is instrumental to modern feed formulation and diet optimization across livestock sectors. Mechanistic models of nutrient digestion, absorption, and metabolism are implemented in industry as ‘decision support systems’, and in academia to summarize and examine our cumulative biological knowledge, identify knowledge gaps and test/evaluate hypotheses. However, comparatively less progress in this field has been made in the equine sector. This limits the ability of the equine sector to address complex challenges such as interactions between equine nutrition, management, health, and welfare. To address this gap, a first iteration of a dynamic, deterministic, mechanistic model describing postabsorptive nutrient dynamics for equines has been developed. Model coding and development was performed using Python with specific libraries for mathematical equations and integration. This model was developed primarily based on extant published mechanistic models of protein and energy metabolism in other species. The present model simulates the partitioning of ingested nutrients into digested nutrients, through intermediary metabolism and ultimately to growth and deposition of protein, fat, water, and ash into the body compartments. As shown in Figure 1, the model takes digested nutrients (protein, fat, starch, glucose, volatile fatty acids) as inputs and contains 12 state variables divided into metabolite pools (amino acids, fatty acids, glucose, glycogen, acetyl-CoA, ATP), and body constituent pools (muscle protein, bone protein, hide protein, viscera protein, body fat, body ash). Ingested feed is partitioned into digested nutrients using digestibility coefficients from extant equine literature, and indigestible nutrients are excreted. Meta-analysis was used to develop empirical equations to predict VFA concentrations in the hindgut. VFA absorption is estimated using Michaelis-Menten kinetics including the pH and hindgut volume, assumed as a constant value for a mature horse Acetyl-CoA, a key intermediary metabolism pool, is produced from amino acid, glucose, and fatty acid catabolism and is consumed for maintenance requirements, which is accounted for in an ATP pool. The ATP pool is a ‘zero pool’, which balances the ATP producing and -consuming reactions in the model, with any remaining ATP balance required being pulled from the acetyl-CoA pool. Turnover of protein and fat is represented by differences in accretion and catabolism. Work is ongoing to evaluate the developed model for model performance using data extracted from the literature. The model will be trained and subsequently evaluated using equine studies in three main areas: growth and body composition, protein turnover and nitrogen balance, and energy metabolism. Developing this model for the equine sector will stimulate and assist in defining future research priorities, inform revisions to the nutrient requirements of horses, increase understanding of metabolic processes as well as related disorders, reduce waste via increased focus on precision feeding and support the development of new products and services to improve equine health.

170 Understanding how zinc and manganese interact with the hormones found in anabolic implants to alter growth of bovine satellite cells

Abstract It has been well established in both in vitro and in vivo models that the hormones found in anabolic implants [estradiol (E2) and trenbolone acetate (TBA)] positively impact skeletal muscle growth. Previous research has demonstrated that treating primary bovine satellite cells (BSC) with E2 or TBA results in increases in proliferation and protein synthesis and decreases in protein degradation, leading to enhanced skeletal muscle growth. This improved skeletal muscle growth occurs through partially defined pathways, but many unknowns remain. More recently, research has emerged demonstrating that strategic supplementation of specific trace minerals (TM) may further augment the positive effects of E2 and TBA on skeletal muscle growth. There are many ways through which the complex signaling pathways induced by the hormones found in anabolic implants may interact with TM to further enhance skeletal muscle growth. Zinc (Zn) and manganese (Mn) are two of the TM that we have recently investigated. Zinc is the most utilized metal in biological processes and supports animal growth through critical roles in the functions of IGF-1, GH, and DNA synthesis. Manganese is an essential TM providing catalytic activity to enzymes involved in protein glycosylation, nitrogen metabolism, and antioxidant capacity. However, additional research is needed to determine if hormones and TM interact to impact growth of skeletal muscle, specifically. As such, the goal of recent research that has been conducted by our group is to utilize an in vitro primary BSC model to determine whether hormones and TM interact to alter differentiation and protein synthesis. Preliminary research from our group assesses the impacts of varying, physiological concentrations of Zn or Mn on differentiation and protein synthesis of primary BSC cultures that have also been treated with or without the hormones found in anabolic implants. These preliminary findings demonstrate that while there seems to be no interaction between Zn and hormones in differentiating BSC, supplemental Zn on the higher end of physiologically relevant concentrations has a positive effect on signaling pathways involved in growth, protein turnover, skeletal muscle differentiation, and mineral transporters. However, it appears that Zn and Mn each interact with hormones to positively augment protein synthesis in primary BSC cultures. A deeper understanding of the relationship these TM and others may have with hormones will allow for more strategic supplementation practices that can potentially improve skeletal muscle growth.

162 The role of functional amino acids in optimizing nutrition and health

Abstract The ability of a diet or an individual protein-containing ingredient to satisfy the indispensable amino acid (IAA) requirements of an individual is a reflection of protein quality. In human nutrition, the concept of PQ is well recognized and widely applied to facilitate guidance on the extensive range of food items we consume. In the pet food industry, the concept of PQ is gaining recognition as a way in which to identify ideal candidate ingredients for diet formulation and our laboratory has recently completed a large data base of PQ estimates of typical ingredients used in the pet food industry to compliment the emerging PQ ingredient data. Our laboratory has also used more advanced methodologies, specifically the IAAO technique, to measure the dietary Phe, Trp, Met, Thr, and Lys requirements for adult dogs of small, medium and large breed dogs. The role of AA beyond support of protein turnover (adults) or protein deposition (growth) is being explored, such as the role of certain AA in behavior, immune function and inflammation. For example, the ratio of tryptophan to the large neutral amino acids of 0.75:1.0 in contrast to 0.46:1.0 resulted in better indices of gut health and may reduce agonistic behaviors both of which are underpinned by improved serotonergic status. The role of the sulfur AA in the development of dilated cardiomyopathy further highlights the role of IAA as sources for secondary metabolites, such as taurine and glutathione in supporting animal health. Indeed, many AA are becoming referred to as functional AA and the requirement of these AA may differ in healthy and challenged animals and deserve attention in dog and cat nutrition as well. Overall, the pattern of AA that defines the requirement of healthy dogs and cats, may not be the pattern that would support the animal under challenging conditions. Research into supporting different physiological challenges and how these may affect amino acid requirements is warranted.

Comparative Proteomic Analysis Reveals the Effect Mechanisms of Glucose on the Biomass and Phenolic Glycoside Esters Synthesis Activity of Candida Parapsilosis ACCC 20221 Whole-Cell Catalyst.

A new Candida parapsilosis ACCC 20221 (C. parapsilosis ACCC 20221) whole-cell catalyst with a high phenolic glycoside esters synthesis activity and large biomass was obtained after culture with glucose. The possible mechanisms were revealed by using comparative proteomics. It found the expression of proteins involved in post-translational modification, protein turnover, and chaperone, and RNA processing and modification was upregulated, which ensured the metabolic balance and accurate translation, correct folding, and post-translational modification of proteins, thus enhancing the production of lipases in C. parapsilosis ACCC 20221 cultured with glucose. Moreover, the glycolysis pathway, tricarboxylic acid cycle, and fatty acids synthesis were enhanced, while the β-oxidation of fatty acids was weakened in C. parapsilosis ACCC 20221 cells cultured with glucose, which led to an increase in energy generation and cell membrane synthesis; thus, large biomass was obtained. In addition, CCE40476.1 and CAC86400.1, which were likely to exert arbutin esters synthesis activity in C. parapsilosis ACCC 20221, were screened, and it was found that vinyl propionate could easily enter the catalytic pocket of CCE40476.1 and form hydrogen bonding interactions with Leu191 and Ser266.

Proteome Dynamics in iPSC-Derived Human Dopaminergic Neurons

Dopaminergic neurons participate in fundamental physiological processes and are the cell type primarily affected in Parkinson's disease. Their analysis is challenging due to the intricate nature of their function, involvement in diverse neurological processes, and heterogeneity and localization in deep brain regions. Consequently, most of the research on the protein dynamics of dopaminergic neurons has been performed in animal cells exvivo. Here we use iPSC-derived human mid-brain-specific dopaminergic neurons to study general features of their proteome biology and provide datasets for protein turnover and dynamics, including a human axonal translatome. We cover the proteome to a depth of 9409 proteins and use dynamic SILAC to measure the half-life of more than 4300 proteins. We report uniform turnover rates of conserved cytosolic protein complexes such as the proteasome and map the variable rates of turnover of the respiratory chain complexes in these cells. We use differential dynamic SILAC labeling in combination with microfluidic devices to analyze local protein synthesis and transport between axons and soma. We report 105 potentially novel axonal markers and detect translocation of 269 proteins between axons and the soma in the time frame of our analysis (120h). Importantly, we provide evidence for local synthesis of 154 proteins in the axon and their retrograde transport to the soma, among them several proteins involved in RNA editing such as ADAR1 and the RNA helicase DHX30, involved in the assembly of mitochondrial ribosomes. Our study provides a workflow and resource for the future applications of quantitative proteomics in iPSC-derived human neurons.

An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

Novel Biomarkers in Early Prediction of Diabetic Nephropathy: A Systematic Review

Diabetes mellitus (DM) is a metabolic illness that is highly prevalent and intricate. Because of the high incidence of diabetic complications and disease mortality, the condition is one of the biggest medical and social issues in the world. Due to its rising prevalence, diabetes mellitus (DM), which raises blood glucose levels, is one of the world's health challenges and is anticipated to impact 495 million people. Diabetic kidney disease, or DKD, is a widespread ailment all over the world. It is the main cause of end-stage kidney disease (ESKD) and one of the most common consequences of diabetes mellitus (DM). Three essential elements play a role in its pathogenesis: the inflammatory, metabolic, and hemodynamic axis. Clinically, DKD is characterized by persistent albuminuria and a gradual reduction in glomerular filtration rate (GFR). These changes, however, are not unique to DKD, emphasizing the necessity to find new biomarkers originating from the disease's pathophysiology to support diagnosis, monitoring, treatment response, and prognosis. Cystatin C, also known as CysC, is a member of the cysteine protein inhibitor family and is a low-molecular-weight protein with 122 amino acids. The CST3 housekeeping gene, which is found on chromosome 20 (20pl1. 2, encodes it. Protein turnover, pro-protein processing, bone remodeling, antigen presentation, and apoptosis are among the physiological processes in which cysteine cathepsins are involved.