Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Neurotensin counteracts hair growth inhibition induced by chronic restraint stress.

Stress has been considered as a potential trigger for hair loss through the neuroendocrine-hair follicle (HF) axis. Neurotensin (NTS), a neuropeptide, is known to be dysregulated in the inflammatory-associated skin diseases. However, the precise role of NTS in stress-induced hair loss is unclear. To investigate the function and potential mechanisms of NTS in stress-induced hair growth inhibition, we initially detected the expression of neurotensin receptor (Ntsr) and NTS in the skin tissues of stressed mice by RNA-sequencing and ELISA. We found chronic restraint stress (CRS) significantly decreased the expression of both NTS and Ntsr in the skin tissues of mice. Intracutaneous injection of NTS effectively counteracted CRS-induced inhibition of hair growth in mice. Furthermore, NTS regulated a total of 1093 genes expression in human dermal papilla cells (HDPC), with 591 genes being up-regulated and 502 genes being down-regulated. GO analysis showed DNA replication, cell cycle, integral component of plasma membrane and angiogenesis-associated genes were significantly regulated by NTS. KEGG enrichment demonstrated that NTS also regulated genes related to the Hippo signalling pathway, axon guidance, cytokine-cytokine receptor interaction and Wnt signalling pathway in HDPC. Our results not only uncovered the potential effects of NTS on stress-induced hair growth inhibition but also provided an understanding of the mechanisms at the gene transcriptional level.

TMTpro-18plex: The Expanded and Complete Set of TMTpro Reagents for Sample Multiplexing.

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

GSK3-like kinases positively modulate abscisic acid signaling through phosphorylating subgroup III SnRK2s in Arabidopsis.

Arabidopsis glycogen synthase kinase 3 (GSK3)-like kinases have versatile functions in plant development and in responding to abiotic stresses. Although physiological evidence suggested a potential role of GSK3-like kinases in abscisic acid (ABA) signaling, the underlying molecular mechanism was largely unknown. Here we identified members of Snf1-related kinase 2s (SnRK2s), SnRK2.2 and SnRK2.3, that can interact with and be phosphorylated by a GSK3-like kinase, brassinosteroid insensitive 2 (BIN2). bin2-3 bil1 bil2, a loss-of-function mutant of BIN2 and its two closest homologs, BIN2 like 1 (BIL1) and BIN2 like 2 (BIL2), was hyposensitive to ABA in primary root inhibition, ABA-responsive gene expression, and phosphorylating ABA Response Element Binding Factor (ABF) 2 fragment by in-gel kinase assays, whereas bin2-1, a gain-of-function mutation of BIN2, was hypersensitive to ABA, suggesting that these GSK3-like kinases function as positive regulators in ABA signaling. Furthermore, BIN2 phosphorylated SnRK2.3 on T180, and SnRK2.3(T180A) had decreased kinase activity in both autophosphorylation and phosphorylating ABFs. Bikinin, a GSK3 kinase inhibitor, inhibited the SnRK2.3 kinase activity and its T180 phosphorylation in vivo. Our genetic analysis further demonstrated that BIN2 regulates ABA signaling downstream of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS receptors and clade A protein phosphatase 2C but relies on SnRK2.2 and SnRK2.3. These findings provide significant insight into the modulation of ABA signaling by Arabidopsis GSK3-like kinases.

γ-Aminobutyric acid promotes the inhibition of hair growth induced by chronic restraint stress.

Stress plays a critical role in hair loss, although the underlying mechanisms are largely unknown. γ-aminobutyric acid (GABA) has been reported to be associated with stress; however, whether it affects stress-induced hair growth inhibition is unclear. This study aimed to investigate the potential roles and mechanisms of action of GABA in chronic restraint stress (CRS)-induced hair growth inhibition. We performed RNA-seq analysis and found that differentially expressed genes (DEGs) associated with neuroactive ligand-receptor interaction, including genes related to GABA receptors, significantly changed after mice were treated with CRS. Targeted metabolomics analysis and enzyme-linked immunosorbent assay (ELISA) also showed that GABA levels in back skin tissues and serum significantly elevated in the CRS group. Notably, CRS-induced hair growth inhibition got aggravated by GABA and alleviated through GABAA antagonists, such as picrotoxin and ginkgolide A. RNA sequencing analysis revealed that DEGs related to the cell cycle, DNA replication, purine metabolism, and pyrimidine metabolism pathways were significantly downregulated in dermal papilla (DP) cells after GABA treatment. Moreover, ginkgolide A, a GABAA antagonist extracted from the leaves of Ginkgo biloba, promoted the cell cycle of DP cells. Therefore, the present study demonstrated that the increase in GABA could promote CRS-induced hair growth inhibition by downregulating the cell cycle of DP cells and suggested that ginkgolide A may be a promising therapeutic drug for hair loss.

The primary signaling outputs of brassinosteroids are regulated by abscisic acid signaling.

Phytohormones have essential roles in coordinately regulating a large array of developmental processes. Studies have revealed that brassinosteroids (BRs) and abscisic acid (ABA) interact to regulate hundreds of expression in genes, governing many biological processes. However, whether their interaction is through modification or intersection of their primary signaling cascades, or by independent or parallel pathways remains a big mystery. Using biochemical and molecular markers of BR signaling and ABA biosynthetic mutants, we demonstrated that exogenous ABA rapidly inhibits BR signaling outputs as indicated by the phosphorylation status of BES1 and BR-responsive gene expression. Experiments using a bri1 null-allele, bri1-116, and analysis of subcellular localization of BKI1-YFP further revealed that the BR receptor complex is not required for ABA to act on BR signaling outputs. However, when the BR downstream signaling component BIN2 is inhibited by LiCl, ABA failed to inhibit BR signaling outputs. Also, using a set of ABA insensitive mutants, we found that regulation of ABA on the BR primary signaling pathway depends on the ABA early signaling components, ABI1 and ABI2. We propose that the signaling cascades of ABA and BR primarily cross-talk after BR perception, but before their transcriptional activation. This model provides a reasonable explanation for why a large proportion of BR-responsive genes are also regulated by ABA, and provides an insight into the molecular mechanisms by which BRs could interact with ABA.

The Mechanisms Underlying PTEN Loss in Human Tumors Suggest Potential Therapeutic Opportunities.

In this review, we will first briefly describe the diverse molecular mechanisms associated with PTEN loss of function in cancer. We will then proceed to discuss the molecular mechanisms linking PTEN loss to PI3K activation and demonstrate how these mechanisms suggest possible therapeutic approaches for patients with PTEN-null tumors.

Molecular Profiling Establishes Genetic Features Predictive of the Efficacy of the p110ß Inhibitor KIN-193.

Aberrant activation of the PI3K pathway is a common alteration in human cancers. Therapeutic intervention targeting the PI3K pathway has achieved limited success due to the intricate balance of its different components and isoforms. Here, we systematically investigated the genomic and transcriptomic signatures associated with response to KIN-193, a compound specifically targeting the p110ß isoform. By integrating genomic, transcriptomic, and drug response profiles from the Genomics of Drug Sensitivity in Cancer database, we identified mutational and transcriptomic signatures associated with KIN-193 and further created statistical models to predict the treatment effect of KIN-193 in cell lines, which may eventually be clinically valuable. These predictions were validated by analysis of the external Cancer Cell Line Encyclopedia dataset. These results may assist precise therapeutic intervention targeting the PI3K pathway. SIGNIFICANCE: These findings provide new insights into molecular signatures associated with sensitivity of the p110ß inhibitor KIN-193, which may provide a useful guide for developing precise treatment methods for cancer.

Abscisic Acid Signaling Inhibits Brassinosteroid Signaling through Dampening the Dephosphorylation of BIN2 by ABI1 and ABI2.

Abscisic acid (ABA) and brassinosteroid (BR) antagonistically regulate many aspects of plant growth and development. Previous physiological studies have revealed that the inhibition of BR signaling by ABA is largely dependent on ABI1 and ABI2. However, the genetic and molecular basis of how ABI1 and ABI2 are involved in inhibiting BR signaling remains unclear. Although it is known that in the BR signaling pathway the ABA-BR crosstalk occurs in the downstream of BR receptor complex but upstream of BIN2 kinase, a negative regulator of BR signaling, the component that acts as the hub to directly mediate their crosstalk remains a big mystery. Here, we found that ABI1 and ABI2 interact with and dephosphorylate BIN2 to regulate its activity toward the phosphorylation of BES1. By in vitro mimicking ABA signal transduction, we found that ABA can promote BIN2 phosphorylation by inhibiting ABI2 through ABA receptors. RNA-sequencing analysis further demonstrated that ABA inhibits BR signaling through the ABA primary signaling components, including its receptors and ABI2, and that ABA and GSK3s co-regulate a common set of stress-responsive genes. Because BIN2 can interact with and phosphorylate SnRK2s to activate its kinase activity, our study also reveals there is a module of PP2Cs-BIN2-SnRK2s in the ABA signaling pathway. Collectively, these findings provide significant insights into how plants balance growth and survival by coordinately regulating the growth-promoting signaling pathway and stress responses under abiotic stresses.

SINAT E3 Ligases Control the Light-Mediated Stability of the Brassinosteroid-Activated Transcription Factor BES1 in Arabidopsis.

The plant hormones brassinosteroids (BRs) participate in light-mediated regulation of plant growth, although the underlying mechanisms are far from being fully understood. In addition, the function of the core transcription factor in the BR signaling pathway, BRI1-EMS-SUPPRESSOR 1 (BES1), largely depends on its phosphorylation status and its protein stability, but the regulation of BES1 is not well understood. Here, we report that SINA of Arabidopsis thaliana (SINATs) specifically interact with dephosphorylated BES1 and mediate its ubiquitination and degradation. Our genetic data demonstrated that SINATs inhibit BR signaling in a BES1-dependent manner. Interestingly, we found that the protein levels of SINATs were decreased in the dark and increased in the light, which changed BES1 protein levels accordingly. Thus, our study not only uncovered a new mechanism of BES1 degradation but also provides significant insights into how light conditionally regulates plant growth through controlling accumulation of different forms of BES1.

Short-term and continuing stresses differentially interplay with multiple hormones to regulate plant survival and growth.

The stress phytohormone, abscisic acid (ABA), plays important roles in facilitating plants to survive and grow well under a wide range of stress conditions. Previous gene expression studies mainly focused on plant responses to short-term ABA treatment, but the effect of sustained ABA treatment and their difference are poorly studied. Here, we treated plants with ABA for 1 h or 9 d, and our genome-wide analysis indicated the differentially regulated genes under the two conditions were tremendously different. We analyzed other hormones' signaling changes by using their whole sets of known responsive genes as reporters and integrating feedback regulation of their biosynthesis. We found that, under short-term ABA treatment, signaling outputs of growth-promoting hormones, brassinosteroids and gibberellins, and a biotic stress-responsive hormone, jasmonic acid, were significantly inhibited, while auxin and ethylene signaling outputs were promoted. However, sustained ABA treatment repressed cytokinin and gibberellin signaling, but stimulated auxin signaling. Using several sets of hormone-related mutants, we found candidates in corresponding hormonal signaling pathways, including receptors or transcription regulators, are essential in responding to ABA. Our findings indicate interactions of ABA-dependent stress signals with hormones at different levels are involved in plants to survive under transient stress and to adapt to continuing stressful environments.