Degradation-Based Protein Profiling: A Case Study of Celastrol.

Natural products, while valuable for drug discovery, encounter limitations like uncertainty in targets and toxicity. As an important active ingredient in traditional Chinese medicine, celastrol exhibits a wide range of biological activities, yet its mechanism remains unclear. In this study, they introduced an innovative "Degradation-based protein profiling (DBPP)" strategy, which combined PROteolysis TArgeting Chimeras (PROTAC) technology with quantitative proteomics and Immunoprecipitation-Mass Spectrometry (IP-MS) techniques, to identify multiple targets of natural products using a toolbox of degraders. Taking celastrol as an example, they successfully identified its known targets, including inhibitor of nuclear factor kappa B kinase subunit beta (IKKß), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3Kα), and cellular inhibitor of PP2A (CIP2A), as well as potential new targets such as checkpoint kinase 1 (CHK1), O-GlcNAcase (OGA), and DNA excision repair protein ERCC-6-like (ERCC6L). Furthermore, the first glycosidase degrader is developed in this work. Finally, by employing a mixed PROTAC toolbox in quantitative proteomics, they also achieved multi-target identification of celastrol, significantly reducing costs while improving efficiency. Taken together, they believe that the DBPP strategy can complement existing target identification strategies, thereby facilitating the rapid advancement of the pharmaceutical field.

Metalloproteinases as Biomarkers and Sociomarkers in Human Health and Disease.

Metalloproteinases (MPs) are zinc-dependent enzymes with proteolytic activity and a variety of functions in the pathophysiology of human diseases. The main objectives of this review are to analyze a specific family of MPs, the matrix metalloproteinases (MMPs), in the most common chronic and complex diseases that affect patients' social lives and to better understand the nature of the associations between MMPs and the psychosocial environment. In accordance with the PRISMA extension for a scoping review, an examination was carried out. A collection of 24 studies was analyzed, focusing on the molecular mechanisms of MMP and their connection to the manifestation of social aspects in human disease. The complexity of the relationship between MMP and social problems is presented via an interdisciplinary approach based on complexity paradigm as a new approach for conceptualizing knowledge in health research. Finally, two implications emerge from the study: first, the psychosocial states of individuals have a profound impact on their overall health and disease conditions, which implies the importance of adopting a holistic perspective on human well-being, encompassing both physical and psychosocial aspects. Second, the use of MPs as biomarkers may provide physicians with valuable tools for a better understanding of disease when used in conjunction with "sociomarkers" to develop mathematical predictive models.

Human colostrum in vitro protein digestion: peptidomics by liquid chromatography-Orbitrap-high-resolution MS and prospection for bioactive peptides via bioinformatics.

Breast milk is known to contain bioactive peptides that are released during digestion, being a major source of bioactive peptides to the new-born, some of which act against invading pathogens. However, the formation of bioactive peptides during digestion of human colostrum remains largely uninvestigated. This study aimed to investigate the formation of peptides during simulated digestion of human colostrum from adult women and to prospect antimicrobial peptides. For this purpose, we used high-resolution MS to monitor the release of peptides during in vitro digestion. Bioinformatics was used for the prospection of antimicrobial activity of peptides. During simulated digestion (oral, gastric and duodenal phases), 2318 peptide sequences derived from 112 precursor proteins were identified. At the end of simulated digestion, casein-derived peptide sequences were the most frequently observed. Among precursors, some proteins were seen for the first time in this study. The resulting peptides were rich in proline, glutamine, valine and leucine residues, providing characteristic traits of antimicrobial peptides. From bioinformatics analysis, seven peptides showed potentially high antimicrobial activity towards bacteria, viruses and fungi, from which the latter was the most prominent predicted activity. Antimicrobial peptides released during digestion may provide a defence platform with controlled release for the new-born.

Recent advances in targeted protein degraders as potential therapeutic agents.

Targeted protein degradation (TPD) technology has gradually become widespread in the past 20 years, which greatly boosts the development of disease treatment. Contrary to small inhibitors that act on protein kinases, transcription factors, ion channels, and other targets they can bind to, targeted protein degraders could target "undruggable targets" and overcome drug resistance through ubiquitin-proteasome pathway (UPP) and lysosome pathway. Nowadays, some bivalent degraders such as proteolysis-targeting chimeras (PROTACs) have aroused great interest in drug discovery, and some of them have successfully advanced into clinical trials. In this review, to better understand the mechanism of degraders, we elucidate the targeted protein degraders according to their action process, relying on the ubiquitin-proteasome system or lysosome pathway. Then, we briefly summarize the study of PROTACs employing different E3 ligases. Subsequently, the effect of protein of interest (POI) ligands, linker, and E3 ligands on PROTAC degradation activity is also discussed in detail. Other novel technologies based on UPP and lysosome pathway have been discussed in this paper such as in-cell click-formed proteolysis-targeting chimeras (CLIPTACs), molecular glues, Antibody-PROTACs (Ab-PROTACs), autophagy-targeting chimeras, and lysosome-targeting chimeras. Based on the introduction of these degradation technologies, we can clearly understand the action process and degradation mechanism of these approaches. From this perspective, it will be convenient to obtain the development status of these drugs, choose appropriate degradation methods to achieve better disease treatment and provide basis for future research and simultaneously distinguish the direction of future research efforts.

Calpain and Cardiometabolic Diseases.

Calpain is defined as a member of the superfamily of cysteine proteases possessing the CysPC motif within the gene. Calpain-1 and -2, which are categorized as conventional isozymes, execute limited proteolysis in a calcium-dependent fashion. Accordingly, the calpain system participates in physiological and pathological phenomena, including cell migration, apoptosis, and synaptic plasticity. Recent investigations have unveiled the contributions of both conventional and unconventional calpains to the pathogenesis of cardiometabolic disorders. In the context of atherosclerosis, overactivation of conventional calpain attenuates the barrier function of vascular endothelial cells and decreases the immunosuppressive effects attributed to lymphatic endothelial cells. In addition, calpain-6 induces aberrant mRNA splicing in macrophages, conferring atheroprone properties. In terms of diabetes, polymorphisms of the calpain-10 gene can modify insulin secretion and glucose disposal. Moreover, conventional calpain reportedly participates in amino acid production from vascular endothelial cells to induce alteration of amino acid composition in the liver microenvironment, thereby facilitating steatohepatitis. Such multifaceted functionality of calpain underscores its potential as a promising candidate for pharmaceutical targets for the treatment of cardiometabolic diseases. Consequently, the present review highlights the pivotal role of calpains in the complications of cardiometabolic diseases and embarks upon a characterization of calpains as molecular targets.

[Effects of branched-chain amino acid supplementation on skeletal muscle proteolytic pathways after exercise-induced muscle injury in rats].

OBJECTIVE: To explore the role of branched-chain amino acid(BCAA) supplementation on muscle damage and the regulation of Krüppel-like factor 15(KLF15) and nuclear factor kappa B(NF-κB) mediated proteolytic pathways after an acute eccentric exercise. METHODS: Male SD rats were divided into placebo group(PLA) and BCAA group(BCAA) randomly, 32 rats per group. Both group were then placed into subgroups: placebo and pre-exercise group(PC), placebo and immediately after exercise group(PE), placebo and 6 h after exercise group(PE6), placebo and 12 h after exercise group(PE12), BCAA and pre-exercise group(BC), BCAA and immediately after exercise group(BE), BCAA and 6 h after exercise group(BE6), BCAA and 12 h after exercise group(BE12), 8 rats per group. Rats in BCAA groups were supplied with BCAA(1 g/(kg·d·BW), 3 days) before the exercise day and placebo groups with equal volume of distilled water. The exercised groups performed a 2 h eccentric exercise on treadmill(16 m/min, -16° slope). Blood and gastrocnemius were collected according to the time points. RT-qPCR was used to measure the mRNA expression of KLF15, NF-κB, FoxO1, Atrogin-1 and MuRF1 in gastrocnemius. RESULTS: (1) No damage was found in myocytes of BC and PC group. The process of morphological damage in BCAA group was relatively faster. (2) The mRNA expression levels of KLF15, FoXO1, Atrogin-1 and MuRF1 in PE were higher than those in PC(P<0.05, P<0.01), NF-κB and Atrogin-1 in PE12 were higher than those in PC(P<0.05). The mRNA expression levels of FoXO1 in BE were higher than those in BC(P<0.05). Compared with PE, the mRNA expression levels of KLF15, Atrogin-1 and MuRF1 in BE were lower(P<0.05, P<0.01), NF-κB and Atrogin-1 in BE12 were lower than those in PE12(P<0.05). The level of serum 3-MH in PE12 group was higher than that in PC group(P<0.05). CONCLUSION: The proteolysis of skeletal muscle after high-intensity eccentric exercise is mediated by two different pathways: KLF15 and NF-κB, whose activation is time-dependent. BCAA may reduce skeletal muscle proteolysis by lowering the level of gene transcription in the KLF15 and NF-κB related protein degradation pathway, which occurs immediately after exercise.

Increasing the Overall Proteome Coverage by Combining Protein Digestion by Tryp-N and Trypsin.

Mass spectrometry-based proteomics combining more than one protease in parallel facilitates the identification of more peptides and proteins than when a single protease is used. Trypsin cleaves proteins C-terminally to arginine and lysine, while its mirroring protease Tryp-N cleaves N-terminally to the same amino acids. Here, we combine trypsin and Tryp-N with the commercially available S-Trap columns, which purify protein samples and catalyze digestion. Comparison of trypsin or Tryp-N coupled with S-Trap columns demonstrates plasma and cell lysate proteins unique to one protease. We thus suggest the use of both proteases in a complementary manner to obtain deeper proteome coverage.

Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Boosting stability and therapeutic potential of proteolysis-resistant antimicrobial peptides by end-tagging ß-naphthylalanine.

Recently, much emphasis has been placed on solving the intrinsic defects of antimicrobial peptides (AMPs), especially their susceptibility to protease digestion for the systemic application of antibacterial biomaterials. Although many strategies have increased the protease stability of AMPs, antimicrobial activity was severely compromised, thereby substantially weakening their therapeutic effect. To address this issue, we introduced hydrophobic group modifications at the N-terminus of proteolysis-resistant AMPs D1 (AArIIlrWrFR) through end-tagging with stretches of natural amino acids (W and I), unnatural amino acid (Nal) and fatty acids. Of these peptides, N1 tagged with a Nal at N-terminus showed the highest selectivity index (GMSI=19.59), with a 6.73-fold improvement over D1. In addition to potent broad-spectrum antimicrobial activity, N1 also exhibited high antimicrobial stability toward salts, serum and proteases in vitro and ideal biocompatibility and therapeutic efficacy in vivo. Furthermore, N1 killed bacteria through multiple mechanisms, involving disruption of bacterial membranes and inhibition of bacterial energy metabolism. Indeed, appropriate terminal hydrophobicity modification opens up new avenues for developing and applying high-stability peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: To improve the potency and stability of proteolysis-resistant antimicrobial peptides (AMPs) without increasing toxicity, we constructed a convenient and tunable platform based on different compositions and lengths of hydrophobic end modifications. By tagging an Nal at the N-terminal, the obtained target compound N1 exhibited strong antimicrobial activity and desirable stability under multifarious environments in vitro (proteases, salts and serum), and also showed favorable biocompatibility and therapeutic efficacy in vivo. Notably, N1 exerted its bactericidal effect by damaging bacterial cell membranes and inhibiting bacterial energy metabolism in a dual mode. The findings provide a potential method for designing or optimizing proteolysis-resistant AMPs thus promoting the development and application of peptide-based antibacterial biomaterial.

γ-Glutamyl-transpeptidase CsGGT2 functions as light-activated theanine hydrolase in tea plant (Camellia sinensis L.).

Theanine is an important secondary metabolite endowing tea with umami taste and health effects. It is essential to explore the metabolic pathway and regulatory mechanism of theanine to improve tea quality. Here, we demonstrated that the expression patterns of CsGGT2 (γ-glutamyl-transpeptidase), participated in theanine synthesis in vitro in our previous research, are significantly different in the aboveground and underground tissues of tea plants and regulated by light. Light up-regulated the expression of CsHY5, directly binding to the promoter of CsGGT2 and acting as an activator of CsGGT2, with a negative correlation with theanine accumulation. The enzyme activity assays and transient expression in Nicotiana benthamiana showed that CsGGT2, acting as bifunctional protein, synthesize and degrade theanine in vitro and in planta. The results of enzyme kinetics, Surface plasmon resonance (SPR) assays and targeted gene-silencing assays showed that CsGGT2 had a higher substrate affinity of theanine than that of ethylamine, and performed a higher theanine degradation catalytic efficiency. Therefore, light mediates the degradation of theanine in different tissues by regulating the expression of the theanine hydrolase CsGGT2 in tea plants, and these results provide new insights into the degradation of theanine mediated by light in tea plants.

Diosgenin inhibits prostate cancer progression by inducing UHRF1 protein degradation.

Prostate cancer (PCa) represents the second cause of cancer death in adult men. Aberrant overexpression of UHRF1 has been reported in several cancer types, and is regarded as a novel drug target for cancer therapy. Nevertheless, no UHRF1-targeted small molecule inhibitor has been testing in clinical trials. Traditional Chinese medicine (TCM) prescriptions have a long history for the treatment of PCa in China, and Chinese herbal extracts are important resources for new drug discovery. In the present study, we first screened the potentially effective components from the commonly used TCMs for PCa treatment in clinic by using network pharmacology together with molecular docking. We identified diosgenin (DSG) as a small molecule natural compound specifically targeting UHRF1 protein. Furthermore, we validated the results by using the wet lab experiments. DSG, by directly binding UHRF1 protein, induced UHRF1 protein degradation through the ubiquitin-proteasome pathway. Importantly, DSG induced UHRF1 protein degradation by reducing the protein interaction with a deubiquitinase USP7. DSG reduced the level of genomic DNA methylation, and elevated the expression of such tumor suppressor genes as p21, p16 and LXN, thereby resulting in cell cycle arrest, cellular senescence and the inhibition of xenograft tumor growth. We here presented the first report that DSG specifically induced UHRF1 protein degradation, thereby revealing a novel anticancer mechanism of DSG. Altogether, this present study provided a promising strategy to discover new molecule-targeted drugs from small-molecule natural products.

Circadian regulation of cardiac muscle function and protein degradation.

The circadian clock plays a fundamental role in physiology. In particular, the heart is a target organ where the clock orchestrates various aspects of cardiac function. At the molecular level, the clock machinery governs daily rhythms of gene expression. Such circadian regulation is in tune with the dynamic nature of heart structure and function, and provides the foundation for chronotherapeutic applications in cardiovascular diseases. In comparison, a regulatory role of the clock in cardiac protein degradation is poorly documented. Sarcomere is the structural and functional unit responsible for cardiac muscle contraction, and sarcomere components are closely regulated by protein folding and proteolysis. Emerging evidence supports a role of the circadian clock in governing sarcomere integrity and function. Particularly, recent studies uncovered a circadian regulation of a core sarcomere component TCAP. It is possible that circadian regulation of the cardiac muscle protein turnover is a key regulatory mechanism underlying cardiac remodeling in response to physiological and environmental stimuli. While the detailed regulatory mechanisms and the molecular links to cardiac (patho)physiology remain to be further studied, therapeutic strategies targeting circadian control in the heart may markedly enhance intervention outcomes against cardiovascular disease.

Short-term exposure to an obesogenic diet causes dynamic dysregulation of proteasome-mediated protein degradation in the hypothalamus of female rats.

OBJECTIVES: Previous work has shown that exposure to a high fat diet dysregulates the protein degradation process in the hypothalamus of male rodents. However, whether this occurs in a sex-independent manner is unknown. The objective of this study was to determine the effects of a short-term obesogenic diet on the ubiquitin-proteasome mediated protein degradation process in the hypothalamus of female rats. METHODS: We fed young adult female rats a high fat diet or standard rat chow for 7 weeks. At the end of the 7th week, animals were euthanized and hypothalamus nuclear and cytoplasmic fractions were collected. Proteasome activity and degradation-specific (K48) ubiquitin signaling were assessed. Additionally, we transfected female rats with CRISPR-dCas9-VP64 plasmids in the hypothalamus prior to exposure to the high fat diet in order to increase proteasome activity and determine the role of reduced proteasome function on weight gain from the obesogenic diet. RESULTS: We found that across the diet period, females gained weight significantly faster on the high fat diet than controls and showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. Notably, while our CRISPR-dCas9 manipulation was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. CONCLUSIONS: Collectively, these results reveal that acute exposure to an obesogenic diet causes reductions in the protein degradation process in the hypothalamus of females.

Design, synthesis, and biological evaluation of novel protopanoxadiol derivatives based PROTACs technology for the treatment of lung cancer.

Protopanoxadiol is a key active ingredient derived from Panax ginseng that is well-known to exhibit anti-tumor activity. Previous research focused on the natural protopanaxadiol derivative AD-1 has demonstrated that it possesses broad spectrum anti-tumor activities in vitro and in vivo. However, its limited activity, selectivity, and cell permeability have impeded its therapeutic application. Herein, a series of novel AD-1 derivatives were designed and synthesized based on proteolysis-targeting chimera (PROTAC) technology by linking AD-1 at the C-3 and C-12 positions with pomalidomide through linkers of alkyl chain of differing lengths to achieve the goal of improving the efficacy of the parent compound. Among these synthesized PROTACs, the representative compound A05 exhibited the most potent anti-proliferative activity against A549 cells. Furthermore, mechanistic studies revealed that compound A05 was able to suppress MDM2 expression, disrupt interactions between p53 and MDM2 and readily induce apoptotic death via the mitochondrial apoptosis pathway. Moreover, the in vivo assays revealed that compound A05 exhibited both anti-proliferative and anti-metastatic activities in the zebrafish tumor xenograft model with A549 cells. Together, our findings suggest that AD-1 based PROTACs associated with the degradation of MDM2 may have promising effects for the treatment of lung cancer and this work provide a foundation for future efforts to develop novel anti-tumor agents from natural products.

Effect of Bear Garlic Addition on the Chemical Composition, Microbiological Quality, Antioxidant Capacity, and Degree of Proteolysis in Soft Rennet Cheeses Produced from Milk of Polish Red and Polish Holstein-Friesian Cows.

This study aimed to assess the effect of milk source and bear garlic addition on the selected properties of soft rennet cheese. Cheeses were produced from cow milk derived from two sources: Polish Red cows (PR) and Polish Holstein-Friesian cows (PHF) with a 0.5% (w/w) addition of bear garlic (Allium ursinum L.) dried leaves. Chemical composition and fatty acid profiles (GC) were determined in fresh cheeses. Fresh and stored for two weeks cheeses were subjected to microbiological studies, i.e., total aerobic bacteria count (TABC); count of Lactococcus sp., yeast and molds; coliforms; analysis of the proteolysis extension by means of o-phthaldialdehyde (OPA) assay and free amino acids content (HPLC); antioxidant capacity as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and ferric reducing antioxidant power (FRAP); as well as pH and water activity. Cheeses with bear garlic herbs were more prone to proteolysis but this was not accompanied by any effect on the microbial counts, water activity or pH. Cheeses produced from PR milk contained less monounsaturated fatty acids (MUFA) but were richer in n-3 PUFA and had a lower n-6/n-3 FA ratio than cheeses from PHF milk. Bear garlic addition increased DPPH anti-radical power but had less of an effect on the FRAP values.

Astaxanthin Inhibits Oxidative Stress-Induced Ku Protein Degradation and Apoptosis in Gastric Epithelial Cells.

Oxidative stress induces DNA damage which can be repaired by DNA repair proteins, such as Ku70/80. Excess reactive oxygen species (ROS) stimulate the activation of caspase-3, which degrades Ku 70/80. Cells with decreased Ku protein levels undergo apoptosis. Astaxanthin exerts antioxidant activity by inducing the expression of catalase, an antioxidant enzyme, in gastric epithelial cells. Therefore, astaxanthin may inhibit oxidative stress-induced DNA damage by preventing Ku protein degradation and thereby suppressing apoptosis. Ku proteins can be degraded via ubiquitination and neddylation which adds ubiquitin-like protein to substrate proteins. We aimed to determine whether oxidative stress decreases Ku70/80 expression through the ubiquitin-proteasome pathway to induce apoptosis and whether astaxanthin inhibits oxidative stress-induced changes in gastric epithelial AGS cells. We induced oxidative stress caused by the treatment of ß-D-glucose (G) and glucose oxidase (GO) in the cells. As a result, the G/GO treatment increased ROS levels, decreased nuclear Ku protein levels and Ku-DNA-binding activity, and induced the ubiquitination of Ku80. G/GO increased the DNA damage marker levels (γ-H2AX; DNA fragmentation) and apoptosis marker annexin V-positive cells and cell death. Astaxanthin inhibited G/GO-induced alterations, including Ku degradation in AGS cells. MLN4924, a neddylation inhibitor, and MG132, a proteasome inhibitor, suppressed G/GO-mediated DNA fragmentation and decreased cell viability. These results indicated that G/GO-induced oxidative stress causes Ku protein loss through the ubiquitin-proteasome pathway, resulting in DNA fragmentation and apoptotic cell death. Astaxanthin inhibited oxidative stress-mediated apoptosis via the reduction of ROS levels and inhibition of Ku protein degradation. In conclusion, dietary astaxanthin supplementation or astaxanthin-rich food consumption may be effective for preventing or delaying oxidative stress-mediated cell damage by suppressing Ku protein loss and apoptosis in gastric epithelial cells.

Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells.

Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37 °C in customized Dulbecco's modified Eagle medium containing 5 mmol/L D-glucose, 1 mmol/L L-phenylalanine, L-[ring-2,4-3H]phenylalanine, 0.1 (control), 0.25, 0.5, 1, or 2 mmol/L L-leucine, L-isoleucine or L-valine or an equimolar mixture of the three BCAAs. The culture medium also contained physiological concentrations of other amino acids found in the plasma of lactating sows. Proliferation, protein synthesis, proteolysis, ß-casein production, the mechanistic target of rapamycin (mTOR) signaling, and the ubiquitin-proteasome pathway were determined for PMECs. Cell proliferation and abundances of phosphorylated mTOR, eukaryotic translation initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase ß-1 proteins increased (P < 0.05), but abundances of ubiquitinated protein and 20S proteasome decreased (P < 0.05) when extracellular concentrations of L-leucine, L-isoleucine, L-valine, or an equimolar mixture of BCAAs were increased from 0.1 to 2 mmol/L. Compared with the control, 0.25, 0.5, 1 or 2 mmol/L BCAAs enhanced (P < 0.01) protein (including ß-casein) synthesis, while decreasing (P < 0.05) proteolysis in PMECs in a dose-dependent manner. Collectively, our results indicate that physiological concentrations of BCAAs regulate protein turnover in mammary epithelial cells to favor net protein synthesis through stimulating the mTOR signaling pathway and inhibiting the ubiquitin-proteasome pathway.

Effect of symbiotic N2 fixation on leaf protein contents, protein degradation and nitrogen resorption during leaf senescence in temperate deciduous woody species.

Nitrogen (N) resorption from senescing leaves enables plants to reuse N, making them less dependent on current N uptake from the environment, leading to higher fitness, particularly under low N supply. Species that form a symbiotic association with N2-fixing bacteria have not evolved proficient N resorption, i.e., they retain more N in the senesced leaves than non-N2-fixing species. However, the physiological mechanism underlying the difference is still unknown. Metabolic and structural protein contents in green and senesced leaves, as well as protein degradation during leaf senescence-a critical initial process for subsequent N resorption-were determined in four N2-fixing legumes and in four non-N2-fixers. The metabolic proteins were highly degraded in legumes and to a lesser extent in nonlegumes. Nonetheless, legumes retained more metabolic proteins in their senesced leaves than nonlegumes, because symbiotic N2 fixation improved the metabolic protein content in green leaves. Symbiotic N2 fixation did not change the structural protein content in green leaves. The structural proteins were moderately degraded in nonlegumes, and almost undegraded in legumes, and more structural proteins remained in the senesced leaves of legumes than in those of nonlegumes. The higher metabolic and structural protein contents in the senesced leaves of N2-fixing legumes properly explained the less proficient N resorption. This is an important step in unraveling molecular mechanisms of different N conservation strategies among plant functional types.