Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence.

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague.

Profiling Glutathionylome in CD38-Mediated Epithelial-Mesenchymal Transition.

Protein S-glutathionylation is an important posttranslational modification that regulates various cellular processes. However, changes in glutathionylome in epithelial-mesenchymal transition (EMT), a crucial cellular process for embryonic development, wound healing, and carcinoma progression and metastasis, have not been fully characterized. Our previous study revealed that CD38 overexpression decreased cellular nicotinamide adenine dinucleotide (NAD+) levels and caused cells to undergo EMT. In the present study, we engineered a cell system in which the glutathione synthetase (GS) mutant was expressed that catalyzed the formation of a glutathione analogue from azido-alanine to profile changes of glutathionylome in CD38-overexpressing cells. We identified 1298 glutathionylated proteins and revealed that proteins with changed glutathionylation levels involved in EMT associated pathways including epithelial adherens junction, actin cytoskeleton, and integrin signaling. Moreover, the glutathionylation level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) was increased in CD38-overexpressing cells. We further demonstrated that glutathionylation of Cys63 residue in 15-PGDH led to decreased enzymatic activity that could promote EMT by increasing prostaglandin E2 (PGE2). Taken together, these results indicate that the clickable glutathione is an effective probe for glutathionylome profiling, and glutathionylation of 15-PGDH on Cys63 inhibits its enzymatic activity to promote EMT.

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.

The different expression of glycogen phosphorylases in renal clear cell renal carcinoma and chromophobe renal carcinoma.

BACKGROUND: The various pathogenesis between Clear cell renal carcinoma (CCRCC) and Chromophobe renal carcinoma (CHRCC) contributes to the different tumor growth rate and metastasis. In this study, we explored the distinct proteomic profiles between these two cancers and found different expression of glycogen phosphorylases in two cancers. METHODS: We explored novel targets by proteomics. Five CCRCC cases and five CHRCC cases were selected for tandem mass tag-labeling liquid chromatography-mass spectroscopy (LC-MS). Gene ontology and KEGG pathway were applied for bioinformatic analysis. Glycogen phosphorylases were detected by Western blotting. RESULTS: CHRCC were younger, more commonly female, and had larger tumors compared to those with CCRCC. 101 differentially expressed proteins (DEPs) in CCRCC and 235 DEPs in CHRCC were detected by LC-MS. It was found that disruption of metabolic pathways, epithelial cell differentiation, and cell response were the common characters for two tumor types. Activation of cell-cell adhesion and oxidation-reduction process stimulate CCRCC growth and epithelial cell differentiation and transferrin transport was involved in CHRCC growth, We also found that oxidative phosphorylation is activated in CHRCC and inhibited in CCRCC. More importantly, we found and confirmed that upregulation of glycogen phosphorylase liver type in CCRCC and glycogen phosphorylase brain type in CHRCC mediated differential glycogenolysis in the two tumor types, which could serve as potential therapeutic targets. CONCLUSION: We found different expression of glycogen phosphorylases in CCRCC and CHRCC by quantitative proteomics, which provides potential therapeutic targets in the future.

Decreased NAD Activates STAT3 and Integrin Pathways to Drive Epithelial-Mesenchymal Transition.

Nicotinamide adenine dinucleotide (NAD) plays an essential role in all aspects of human life. NAD levels decrease as humans age, and supplementation with NAD precursors plays a protective role against aging and associated disease. Less is known about the effects of decreased NAD on cellular processes, which is the basis for understanding the relationship between cellular NAD levels and aging-associated disease. In the present study, cellular NAD levels were decreased by overexpression of CD38, a NAD hydrolase, or by treating cells with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Quantitative proteomics revealed that declining NAD levels downregulated proteins associated with primary metabolism and suppressed cell growth in culture and nude mice. Decreased glutathione synthesis caused a 4-fold increase in cellular reactive oxygen species levels, and more importantly upregulated proteins related to movement and adhesion. In turn, this significantly changed cell morphology and caused cells to undergo epithelial to mesenchymal transition (EMT). Secretomic analysis also showed that decreased NAD triggered interleukin-6 and transforming growth factor beta (TGFß) secretion, which activated integrin-ß-catenin, TGFß-MAPK, and inflammation signaling pathways to sustain the signaling required for EMT. We further revealed that decreased NAD inactivated sirtuin 1, resulting in increased signal transducer and activator of transcription 3 (STAT3) acetylation and phosphorylation, and STAT3 activation. Repletion of nicotinamide or nicotinic acid inactivated STAT3 and reversed EMT, as did STAT3 inhibition. Taken together, these results indicate that decreased NAD activates multiple signaling pathways to promote EMT and suggests that age-dependent decreases in NAD may contribute to tumor progression. Consequently, repletion of NAD precursors has potential benefits for inhibiting cancer progression.

CA9 Silencing Promotes Mitochondrial Biogenesis, Increases Putrescine Toxicity and Decreases Cell Motility to Suppress ccRCC Progression.

Carbonic anhydrase IX (CA9), a pH-regulating transmembrane protein, is highly expressed in solid tumors, and particularly in clear cell renal cell carcinoma (ccRCC). The catalytic mechanisms of CA9 are well defined, but its roles in mediating cell migration/invasion and survival in ccRCC remain to be determined. Here, we confirmed that the mRNA expression of CA9 in ccRCC was significantly higher than that in para-carcinoma tissues from analysis of the datasets in The Cancer Genome Atlas. CA9 knockdown upregulated oxidative phosphorylation-associated proteins and increased mitochondrial biogenesis, resulting in the reversal of the Warburg phenotype and the inhibition of cell growth. Our study revealed that CA9 knockdown upregulated mitochondrial arginase 2 (ARG2), leading to the accumulation of putrescine, which suppressed ccRCC proliferation. Surfaceomics analysis revealed that CA9 knockdown downregulated proteins associated with extracellular matrix (ECM)-receptor interaction and cell adhesion, resulting in decreased cell migration. CA9 silencing also downregulated amino acid transporters, leading to reduced cellular amino acids. Collectively, our data show that CA9 knockdown suppresses proliferation via metabolic reprogramming and reduced cell migration, reaffirming that CA9 is a potential therapeutic target for ccRCC treatment.

Ancient DNA provides new insight into the maternal lineages and domestication of Chinese donkeys.

BACKGROUND: The donkey (Equus asinus) is an important domestic animal that provides a reliable source of protein and method of transportation for many human populations. However, the process of domestication and the dispersal routes of the Chinese donkey are still unclear, as donkey remains are sparse in the archaeological record and often confused with horse remains. To explore the maternal origins and dispersal route of Chinese donkeys, both mitochondrial DNA D-loop and cytochrome b gene fragments of 21 suspected donkey remains from four archaeological sites in China were amplified and sequenced. RESULTS: Molecular methods of species identification show that 17 specimens were donkeys and three samples had the maternal genetic signature of horses. One sample that dates to about 20,000 years before present failed to amplify. In this study, the phylogenetic analysis reveals that ancient Chinese donkeys have high mitochondrial DNA diversity and two distinct mitochondrial maternal lineages, known as the Somali and Nubian lineages. These results indicate that the maternal origin of Chinese domestic donkeys was probably related to the African wild ass, which includes the Nubian wild ass (Equus africanus africanus) and the Somali wild ass (Equus africanus somaliensis). Combined with historical records, the results of this study implied that domestic donkeys spread into west and north China before the emergence of the Han dynasty. The number of Chinese domestic donkeys had increased primarily to meet demand for the expansion of trade, and they were likely used as commodities or for shipping goods along the Silk Road during the Tang Dynasty, when the Silk Road reached its golden age. CONCLUSIONS: This study is the first to provide valuable ancient animal DNA evidence for early trade between African and Asian populations. The ancient DNA analysis of Chinese donkeys also sheds light on the dynamic process of the maternal origin, domestication, and dispersal route of ancient Chinese donkeys.

Proteomic profiling of IgA nephropathy reveals distinct molecular prognostic subtypes.

IgA nephropathy (IgAN) is a heterogeneous disease, which poses a series of challenges to accurate diagnosis and personalized therapy. Herein, we constructed a systematic quantitative proteome atlas from 59 IgAN and 19 normal control donors. Consensus sub-clustering of proteomic profiles divided IgAN into three subtypes (IgAN-C1, C2, and C3). IgAN-C2 had similar proteome expression patterns with normal control, while IgAN-C1/C3 exhibited higher level of complement activation, more severe mitochondrial injury, and significant extracellular matrix accumulation. Interestingly, the complement mitochondrial extracellular matrix (CME) pathway enrichment score achieved a high diagnostic power to distinguish IgAN-C2 from IgAN-C1/C3 (AUC>0.9). In addition, the proteins related to mesangial cells, endothelial cells, and tubular interstitial fibrosis were highly expressed in IgAN-C1/C3. Most critically, IgAN-C1/C3 had a worse prognosis compared to IgAN-C2 (30% eGFR decline, p = 0.02). Altogether, we proposed a molecular subtyping and prognostic system which could help to understand IgAN heterogeneity and improve the treatment in the clinic.

Dual-pharmacophore artezomibs hijack the Plasmodium ubiquitin-proteasome system to kill malaria parasites while overcoming drug resistance.

Artemisinins (ART) are critical anti-malarials and despite their use in combination therapy, ART-resistant Plasmodium falciparum is spreading globally. To counter ART resistance, we designed artezomibs (ATZs), molecules that link an ART with a proteasome inhibitor (PI) via a non-labile amide bond and hijack parasite's own ubiquitin-proteasome system to create novel anti-malarials in situ. Upon activation of the ART moiety, ATZs covalently attach to and damage multiple parasite proteins, marking them for proteasomal degradation. When damaged proteins enter the proteasome, their attached PIs inhibit protease function, potentiating the parasiticidal action of ART and overcoming ART resistance. Binding of the PI moiety to the proteasome active site is enhanced by distal interactions of the extended attached peptides, providing a mechanism to overcome PI resistance. ATZs have an extra mode of action beyond that of each component, thereby overcoming resistance to both components, while avoiding transient monotherapy seen when individual agents have disparate pharmacokinetic profiles.

Nicotinamide Mononucleotide Administration Amends Protein Acetylome of Aged Mouse Liver.

It is known that the activities of nicotine adenine dinucleotide (NAD+)-dependent deacetylase decline in the aging mouse liver, and nicotinamide mononucleotide (NMN)-mediated activation of deacetylase has been shown to increase healthspans. However, age-induced changes of the acetylomic landscape and effects of NMN treatment on protein acetylation have not been reported. Here, we performed immunoprecipitation coupled with label-free quantitative LC-MS/MS (IPMS) to identify the acetylome and investigate the effects of aging and NMN on liver protein acetylation. In total, 7773 acetylated peptides assigned to 1997 proteins were commonly identified from young and aged livers treated with vehicle or NMN. The major biological processes associated with proteins exhibiting increased acetylation from aged livers were oxidation-reduction and metabolic processes. Proteins with decreased acetylation from aged livers mostly participated in transport and translation processes. Furthermore, NMN treatment inhibited the aging-related increase of acetylation on proteins regulating fatty acid ß oxidation, the tricarboxylic acid (TCA) cycle and valine degradation. In particular, NAD (P) transhydrogenase (NNT) was markedly hyperacetylated at K70 in aged livers, and NMN treatment decreased acetylation intensity without altering protein levels. Acetylation at cytochrome 3a25 (Cyp3a25) at K141 was also greatly increased in aged livers, and NMN treatment totally arrested this increase. Our extensive identification and analysis provide novel insight and potential targets to combat aging and aging-related functional decline.

High VHL Expression Reverses Warburg Phenotype and Enhances Immunogenicity in Kidney Tumor Cells.

Clear cell renal cell carcinoma (ccRCC) is a frequently occurring renal cancer. The Von Hippel-Lindau disease tumor suppressor VHL, a known tumor suppressor gene, is frequently mutated in about 50% of patients with ccRCC. However, it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL. In the present study, we found that higher expression of VHL was correlated with the better disease-free survival (DFS) in ccRCC patients using The Cancer Genome Atlas (TCGA) datasets. We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition (EMT), sterol regulatory element-binding protein 1 (SREBP1)-regulated triglyceride synthesis, and cell proliferation. Proteomic analysis provided us a global view that VHL regulated four biological processes, including metabolism, immune regulation, apoptosis, and cell movement. Importantly, we found that VHL overexpression led to up-regulated expression of proteins associated with antigen processing and interferon-responsive proteins, thus rendering ccRCC cells more sensitive to interferon treatment. We defined an interferon-responsive signature (IRS) composed of ten interferon-responsive proteins, whose mRNA expression levels were positively correlated with DFS in ccRCC patients. Taken together, our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.

Nicotinamide N-Methyltransferase Remodeled Cell Metabolism and Aggravated Proinflammatory Responses by Activating STAT3/IL1ß/PGE2 Pathway.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic methyltransferase, catalyzing N-methylation of nicotinamide (NAM) to form 1-methylnicotinamide (1-MNAM), in which S-adenosyl-l-methionine (SAM) is the methyl donor. It has been well documented that NNMT is elevated in multiple cancers and promotes tumor aggressiveness. In the present study, we investigated the effects of NNMT overexpression on cellular metabolism and proinflammatory responses. We found that NNMT overexpression reduced NAD+ and SAM levels, and activated the STAT3 signaling pathway. Consequently, STAT3 activation upregulated interleukin 1ß (IL1ß) and cyclooxygenase-2 (COX2), leading to prostaglandin E2 (PGE2) accumulation. On the other hand, NNMT downregulated 15-hydroxyprostaglandin dehydrogenase (15-PGDH) which catalyzes PGE2 into inactive molecules. Moreover, secretomic data indicated that NNMT promoted secretion of collagens, pro-inflammatory cytokines, and extracellular matrix proteins, confirming NNMT aggravated inflammatory responses to promote cell growth, migration, epithelial-mesenchymal transition (EMT), and chemoresistance. Taken together, we showed that NNMT played a pro-inflammatory role in cancer cells by activating the STAT3/IL1ß/PGE2 axis and proposed that NNMT was a potential therapeutic target for cancer treatment.

Mechanical force-promoted osteoclastic differentiation via periodontal ligament stem cell exosomal protein ANXA3.

Exosomes play a critical role in intracellular communication. The biogenesis and function of exosomes are regulated by multiple biochemical factors. In the present study, we find that mechanical force promotes the biogenesis of exosomes derived from periodontal ligament stem cells (PDLSCs) and alters the exosomal proteome profile to induce osteoclastic differentiation. Mechanistically, mechanical force increases the level of exosomal proteins, especially annexin A3 (ANXA3), which facilitates exosome internalization to activate extracellular signal-regulated kinase (ERK), thus inducing osteoclast differentiation. Moreover, the infusion of exosomes derived from PDLSCs into mice promotes mechanical force-induced tooth movement and increases osteoclasts in the periodontal ligament. Collectively, this study demonstrates that mechanical force treatment promotes the biogenesis of exosomes from PDLSCs and increases exosomal protein ANXA3 to facilitate exosome internalization, which activates ERK phosphorylation, thus inducing osteoclast differentiation. Our findings shed light on new mechanisms for how mechanical force regulates the biology of exosomes and bone metabolism.

HSP60-knockdown suppresses proliferation in colorectal cancer cells via activating the adenine/AMPK/mTOR signaling pathway.

Colorectal cancer (CRC) is the fourth most lethal cancer in the world. Heat shock protein 60 (HSP60), a mitochondrial chaperone that maintains mitochondrial proteostasis, is highly expressed in tumors compared with in paracancerous tissues, suggesting that high HSP60 expression benefits tumor growth. To determine the effects of HSP60 expression on tumor progression, stable HSP60-knockdown HCT116 cells were constructed in the present study, revealing that knockdown of HSP60 inhibited cell proliferation. Proteomic analysis demonstrated that mitochondrial proteins were downregulated, indicating that knockdown of HSP60 disrupted mitochondrial homeostasis. Metabolomic analysis demonstrated that cellular adenine levels were >30-fold higher in HSP60-knockdown cells than in control cells. It was further confirmed that elevated adenine activated the AMPK signaling pathway, which inhibited mTOR-regulated protein synthesis to slow down cell proliferation. Overall, the current results provide a valuable resource for understanding mitochondrial function in CRC, suggesting that HSP60 may be a potential target for CRC intervention.

Text Mining and Hub Gene Network Analysis of Endometriosis.

This study is aimed at systematically characterizing the endometriosis-associated genes based on text mining and at annotating the functions, pathways, and networks of endometriosis-associated hub genes. We extracted endometriosis-associated abstracts published between 1970 and 2020 from the PubMed database. A neural-named entity recognition and multitype normalization tool for biomedical text mining was used to recognize and normalize the genes and proteins embedded in the abstracts. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to annotate the functions and pathways of recognized genes. Protein-protein interaction analysis was conducted on the genes significantly cooccurring with endometriosis to identify the endometriosis-associated hub genes. A total of 433 genes were recognized as endometriosis-associated genes (P < 0.05), and 154 pathways were significantly enriched (P < 0.05). A network of endometriosis-associated genes with 278 gene nodes and 987 interaction links was established. The 15 proteins that interacted with 20 or more other proteins were identified as the hub proteins of the endometriosis-associated protein network. This study provides novel insights into the hub genes that play key roles in the development of endometriosis and have implications for developing targeted interventions for endometriosis.

Flavonoid 4,4'-dimethoxychalcone suppresses cell proliferation via dehydrogenase inhibition and oxidative stress aggravation.

Flavonoids are natural polyphenolic compounds with a diverse array of biological activities and health-promoting effects. Recent studies have found that 4,4'-dimethoxychalcone (DMC) promoted longevity via autophagy; however, its targets are currently unknown. Herein, we employed an unbiased thermal proteome profiling (TPP) method and identified multiple targets of DMC, including ALDH1A3, ALDH2, and PTGES2. We further determined the dissociation constant (Kd) of DMC and ALDH1A3 to be 2.8 µM using microscale thermophoresis (MST) analysis, which indicated that DMC inhibited ALDH1A3 activity and aggravated cellular oxidative stress. DMC treatment significantly increased cellular reactive oxygen species (ROS) production and inhibited cancer cell growth. Quantitative proteomic analysis showed that DMC upregulated proteins associated with stress-responses and downregulated proteins associated with cell cycle progression, and this was confirmed using cell cycle analysis. Taken together, we showed that TPP is an effective tool with which to identify flavonoid targets and set a precedent for deciphering flavonoid function in the future. We have demonstrated that DMC inhibited cell proliferation via ROS-induced cell cycle arrest and is an anti-proliferative agent in cancer treatment.

Structural Alternation in Heat Shock Proteins of Activated Macrophages.

The inflammatory response of macrophages is an orderly and complex process under strict regulation accompanied by drastic changes in morphology and functions. It is predicted that proteins will undergo structural changes during these finely regulated processes. However, changes in structural proteome in macrophages during the inflammatory response remain poorly characterized. In the present study, we applied limited proteolysis coupled mass spectrometry (LiP-MS) to identify proteome-wide structural changes in lipopolysaccharide (LPS)-activated macrophages. We identified 386 structure-specific proteolytic fingerprints from 230 proteins. Using the Gene Ontology (GO) biological process enrichment, we discovered that proteins with altered structures were enriched into protein folding-related terms, in which HSP60 was ranked as the most changed protein. We verified the structural changes in HSP60 by using cellular thermal shift assay (CETSA) and native CETSA. Our results showed that the thermal stability of HSP60 was enhanced in activated macrophages and formed an HSP10-less complex. In conclusion, we demonstrate that in situ structural systems biology is an effective method to characterize proteomic structural changes and reveal that the structures of chaperone proteins vary significantly during macrophage activation.

Structural basis for the multi-activity factor Rad5 in replication stress tolerance.

The yeast protein Rad5 and its orthologs in other eukaryotes promote replication stress tolerance and cell survival using their multiple activities, including ubiquitin ligase, replication fork remodeling and DNA lesion targeting activities. Here, we present the crystal structure of a nearly full-length Rad5 protein. The structure shows three distinct, but well-connected, domains required for Rad5's activities. The spatial arrangement of these domains suggest that different domains can have autonomous activities but also undergo intrinsic coordination. Moreover, our structural, biochemical and cellular studies demonstrate that Rad5's HIRAN domain mediates interactions with the DNA metabolism maestro factor PCNA and contributes to its poly-ubiquitination, binds to DNA and contributes to the Rad5-catalyzed replication fork regression, defining a new type of HIRAN domains with multiple activities. Our work provides a framework to understand how Rad5 integrates its various activities in replication stress tolerance.

Comparative proteomic analysis identifies biomarkers for renal aging.

Proteomics have long been applied into characterization of molecular signatures in aging. Due to different methods and instrumentations employed for proteomic analysis, inter-dataset validation needs to be performed to identify potential biomarkers for aging. In this study, we used comparative proteomics analysis to profile age-associated changes in proteome and glutathionylome in mouse kidneys. We identified 108 proteins that were differentially expressed in young and aged mouse kidneys in three different datasets; from these, 27 proteins were identified as potential renal aging biomarkers, including phosphoenolpyruvate carboxykinase (Pck1), CD5 antigen-like protein (Cd5l), aldehyde dehydrogenase 1 (Aldh1a1), and uromodulin. Our results also showed that peroxisomal proteins were significantly downregulated in aged mice, whereas IgGs were upregulated, suggesting that peroxisome deterioration might be a hallmark for renal aging. Glutathionylome analysis demonstrated that downregulation of catalase and glutaredoxin-1 (Glrx1) significantly increased protein glutathionylation in aged mice. In addition, nicotinamide mononucleotide (NMN) administration significantly increased the number of peroxisomes in aged mouse kidneys, indicating that NMN enhanced peroxisome biogenesis, and suggesting that it might be beneficial to reduce kidney injuries. Together, our data identify novel potential biomarkers for renal aging, and provide a valuable resource for understanding the age-associated changes in kidneys.

O2-Tuned Protein Synthesis Machinery in Escherichia coli-Based Cell-Free System.

Involved in most aerobic biochemical processes, oxygen affects cellular functions, and organism behaviors. Protein synthesis, as the underlying biological process, is unavoidably affected by the regulation of oxygen delivery and utilization. Bypassing the cell wall, cell-free protein synthesis (CFPS) systems are well adopted for the precise oxygen regulation analysis of bioprocesses. Here a reliable flow platform was developed for measuring and analyzing the oxygen regulation on the protein synthesis processes by combining Escherichia coli-based CFPS systems and a tube-in-tube reactor. This platform allows protein synthesis reactions conducted in precisely controlled oxygen concentrations. For analysis of the intrinsic role of oxygen in protein synthesis, O2-tuned CFPS systems were explored with transcription-translation related parameters (transcripts, energy, reactive oxygen species, and proteomic pathway analysis). It was found that 2% of oxygen was the minimum requirement for protein synthesis. There was translation-related protein degradation in the high oxygen condition leading to a reduction. By combining the precise gas level controlling and open biosystems, this platform is also potential for fundamental understanding and clinical applications by diverse gas regulation in biological processes.