Currently, the bottom-up approach, in which proteins are digested by enzymes such as trypsin prior to mass spectrometry, is the mainstream approach in mass spectrometer-based proteomics. In this ...approach, the enzymatic digestion process strongly affects the reproducibility of protein identification and quantification. Here, we quantitatively evaluated the enzymatic digestion of proteins under various conditions by quantitative proteomics using data-independent acquisition and found that proteins precipitated with acetone after solubilization with SDS were fully digestible without re-solubilization. This result implies that organic solvent treatment makes cells amenable to trypsin digestion. Direct trypsin digestion of methanol-fixed cells achieved the same digestion efficiency and quantitative reproducibility as the conventional method. Furthermore, this method was found to be equally applicable to mouse liver samples. The establishment of this method indicates that the sample preparation process in bottom-up proteomics can be simplified while maintaining high digestion efficiency and is expected to become a general method for sample preparation in bottom-up proteomics in the future.
Purpose To characterize the foot arch height, toe flexor strength, and dynamic balance ability of collegiate female dancers and age-matched non-dancers. Participants and Methods This study included ...20 healthy college-aged female dancers (21.6 ± 0.8 years) and 20 age-matched females (19.7 ± 1.0 years) with no previous experience in sports as non-dancers. Foot arch height was determined by measuring the height of the navicular tuberosity in the standing position using a ruler. Toe flexor strength was measured while seated on a chair using a toe grip dynamometer. Dynamic balance ability was evaluated based on the reach distance measured using a professional Y-balance test kit. Results The collegiate dancers had higher foot arches, greater toe flexor strength, and longer Y-balance test reach distance than the non-dancers. Conclusion The foot arch height, toe flexor strength, and dynamic balance ability of collegiate female dancers were adapted through years of training and were superior to those of non-dancers.
Removal of senescent cells (senolysis) has been proposed to be beneficial for improving age-associated pathologies, but the molecular pathways for such senolytic activity have not yet emerged. Here, ...we identified glutaminase 1 (
) as an essential gene for the survival of human senescent cells. The intracellular pH in senescent cells was lowered by lysosomal membrane damage, and this lowered pH induced kidney-type glutaminase (KGA) expression. The resulting enhanced glutaminolysis induced ammonia production, which neutralized the lower pH and improved survival of the senescent cells. Inhibition of KGA-dependent glutaminolysis in aged mice eliminated senescent cells specifically and ameliorated age-associated organ dysfunction. Our results suggest that senescent cells rely on glutaminolysis, and its inhibition offers a promising strategy for inducing senolysis in vivo.
C-C chemokine ligand 2 (CCL2) plays pivotal roles in tumor formation, progression, and metastasis. Although CCL2 expression has been found to be dependent on the nuclear factor (NF)-κB signaling ...pathway, the regulation of CCL2 production in tumor cells has remained unclear. We have identified a noncanonical pathway for regulation of CCL2 production that is mediated by mammalian target of rapamycin complex 1 (mTORC1) but independent of NF-κB. Multiple phosphoproteomics approaches identified the transcription factor forkhead box K1 (FOXK1) as a downstream target of mTORC1. Activation of mTORC1 induces dephosphorylation of FOXK1, resulting in transactivation of the CCL2 gene. Inhibition of the mTORC1-FOXK1 axis attenuated insulin-induced CCL2 production as well as the accumulation of tumor-associated monocytes-macrophages and tumor progression in mice. Our results suggest that FOXK1 directly links mTORC1 signaling and CCL2 expression in a manner independent of NF-κB and that CCL2 produced by this pathway contributes to tumor progression.
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•mTORC1 regulates CCL2 expression via FOXK1 activation•FOXK1 is dephosphorylated and thereby activated in response to mTORC1 activation•The mTORC1-FOXK1-CCL2 pathway is independent of classical NF-κB signaling•This pathway promotes macrophage accumulation and associated tumor growth
Nakatsumi et al. show that mTORC1 regulates CCL2 expression in a manner independent of NF-κB signaling by dephosphorylating the transcription factor FOXK1. Moreover, they demonstrate that hyperactivation of mTORC1 results in attraction of M2-type tumor-associated macrophages and promotes tumor growth in vivo via the mTORC1-FOXK1-CCL2 pathway.
Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by ...large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment.
Mechanistic target of rapamycin complex 1 (mTORC1) is a serine-threonine kinase that is activated by extracellular signals, such as nutrients and growth factors. It plays a key role in the control of ...various biological processes, such as protein synthesis and energy metabolism by mediating or regulating the phosphorylation of multiple target molecules, some of which remain to be identified. We have here reanalysed a large-scale phosphoproteomics data set for mTORC1 target molecules and identified pre-B cell leukemia transcription factor 2 (PBX2) as such a novel target that is dephosphorylated downstream of mTORC1. We confirmed that PBX2, but not other members of the PBX family, is dephosphorylated in an mTORC1 activity-dependent manner. Furthermore, pharmacological and gene knockdown experiments revealed that glycogen synthase kinase 3 (GSK3) and protein phosphatase 1 (PP1) are responsible for the phosphorylation and dephosphorylation of PBX2, respectively. Our results thus suggest that the balance between the antagonistic actions of GSK3 and PP1 determines the phosphorylation status of PBX2 and its regulation by mTORC1.
Background & Aims Small nucleolar noncoding RNAs (snoRNAs) regulate function of ribosomes, and specific snoRNAs are dysregulated in some cancer cells. We investigated dysregulation of snoRNAs in ...pancreatic ductal adenocarcinoma (PDAC) cells. Methods We investigated snoRNA expression in PDAC cell lines by complementary DNA microarray and quantitative reverse transcription polymerase chain reaction. In PDAC (n = 133), intraductal papillary mucinous neoplasm (n = 16), mucinous cystic neoplasm-associated PDAC (n = 1), and non-tumor pancreas (n = 8) and liver (n = 3) tissues from subjects who underwent surgical resection, levels of snoRNA were measured by quantitative reverse transcription polymerase chain reaction and compared with clinicopathologic parameters and survival times determined by Kaplan−Meier analysis. To examine snoRNA function, PDAC cells were transfected with snoRNA-antisense oligonucleotides flanked with amido-bridged nucleic acids, or snoRNA-expression plasmids, and analyzed in proliferation, colony formation, spheroid formation, and invasion assays. To identify snoRNA-related factors, cells were analyzed by gene expression and proteomic profiling and immunoblot assays. Mice were given intrasplenic injections of MIA PaCa2- or Suit2-HLMC cells; tumor-bearing nude mice were then given 3 weekly injections of an antisense oligonucleotides against SNORA23, a H/ACA-box type snoRNA, and tumor growth and metastasis to liver, blood, and pancreas were analyzed. Results Levels of SNORA23 increased and accumulated at the nucleolus in highly metastatic MIA PaCa2- or Suit2-HLMC cells compared with their parental cells. We detected SNORA23 in human PDAC specimens but not in non-tumor pancreatic tissue. PDAC level of SNORA23 correlated with invasion grade and correlated inversely with disease-free survival time of patients. Expression of SNORA23 in PDAC cells increased their invasive activity and colony formation, and spheroid formation was inhibited by SNORA23 knockdown. In gene expression and proteomic profile analyses, we found SNORA23 to increase expression of spectrin repeat-containing nuclear envelope 2 (SYNE2) messenger RNA and protein. Knockdown of SYNE2 in PDAC cells reduced their invasive activities and anchor-independent survival. Administration of SNORA23 antisense oligonucleotides to mice slowed growth of xenograft tumors, tumor expression of SYNE2, tumor cell dissemination, and metastasis to liver. Conclusions We found expression of the snoRNA SNORA23, which mediates sequence-specific pseudouridylation of ribosomal RNAs, to be increased in human PDAC tissues compared with non-tumor tissues, and levels to correlate with tumor invasion grade and patient survival time. SNORA23 increases expression of SYNE2, possibly through modulation of ribosome biogenesis, to promote PDAC cell survival and invasion, and growth and metastasis of xenograft tumors in mice.
Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show ...that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.
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•PKM1 promotes tumor growth cell intrinsically in some contexts•PKM1 activates glucose catabolism without interfering with biosynthetic pathways•PKM1-dependent autophagy/mitophagy contributes to malignancy•Expression of PKM1, but not PKM2, is sufficient to support SCLC cell proliferation
The relative importance of PKM isoforms in tumor growth has been controversial. Morita et al. show that PKM1 promotes the growth of multiple tumor models using mouse lines expressing PKM1 or PKM2 from the endogenous Pkm locus. PKM1 is expressed in human SCLC, and it is important for SCLC cell proliferation.
Proteomics is a powerful tool for obtaining information on a large number of proteins with regard to their expression levels, interactions with other molecules, and posttranslational modifications. ...Whereas nontargeted, discovery proteomics uncovers differences in the proteomic landscape under different conditions, targeted proteomics has been developed to overcome the limitations of this approach with regard to quantitation. In addition to technical advances in instruments and informatics tools, the advent of the synthetic proteome composed of synthetic peptides or recombinant proteins has advanced the adoption of targeted proteomics across a wide range of research fields. Targeted proteomics can now be applied to measurement of the dynamics of any proteins of interest under a variety of conditions as well as to estimation of the absolute abundance or stoichiometry of proteins in a given network. Multiplexed targeted proteomics assays of high reproducibility and accuracy can provide insight at the quantitative level into entire networks that govern biological phenomena or diseases. Such assays will establish a new paradigm for data-driven science.
In the mammalian circadian clockwork, CRY1 and CRY2 repressor proteins are regulated by posttranslational modifications for temporally coordinated transcription of clock genes. Previous studies ...revealed that FBXL3, an F-box-type E3 ligase, ubiquitinates CRYs and mediates their degradation. Here, we found that FBXL21 also ubiquitinates CRYs but counteracts FBXL3. Fbxl21−/− mice exhibited normal periodicity of wheel-running rhythms with compromised organization of daily activities, while an extremely long-period phenotype of Fbxl3−/− mice was attenuated in Fbxl3/Fbxl21 double-knockout mice. The double knockout destabilized the behavioral rhythms progressively and sometimes elicited arrhythmicity. Surprisingly, FBXL21 stabilized CRYs and antagonized the destabilizing action by FBXL3. Predominantly cytosolic distribution of FBXL21 contrasts with nuclear localization of FBXL3. These results emphasize the physiological importance of antagonizing actions between FBXL21 and FBXL3 on CRYs, and their combined actions at different subcellular locations stabilize oscillation of the circadian clock.
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► FBXL21 stabilizes CRY1 and CRY2, key players in the circadian clockwork ► FBXL21 action antagonizes the destabilizing action of FBXL3 on CRYs ► Depletion of Fbxl21/Fbxl 3 destabilized behavioral rhythm or caused arrhythmicity ► FBXL21 and FBXL3 actions are vital for robustness of the circadian clock oscillation.
FBXL21, an F-box-type ubiquitin E3 ligase, is identified as a regulator of the circadian clock repressor CRYPTOCHROME (CRY) proteins. FBXL21 has the opposite effects on circadian period and CRY stability compared to its homolog, FBXL3, and their combined actions at different subcellular locations ensures stable oscillation of the circadian clock.
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