Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response. Antioxid. Redox Signal. 19, 998-1011.
SummaryThe recessive Arabidopsis thaliana fumonisin B1-resistant (fbr6) mutant was identified by its ability to survive in the presence of a programmed cell death (PCD)-inducing fungal toxin FB1. The fbr6 mutant also displays altered plant architecture in the absence of FB1, most notably elongated petioles and enhanced leaf margin serration. These phenotypes are a result of a T-DNA insertion in the SQUAMOSA promoter binding protein (SBP) domain gene, AtSPL14. AtSPL14 encodes a plant-specific protein with features characteristic of a transcriptional regulator, including a nuclear localization signal sequence, a plant-specific DNA binding domain (the SBP box), and a protein interaction motif (ankyrin repeats). A transiently expressed fusion of the AtSPL14 protein to green fluorescent protein is directed to the plant nucleus. DNA sequences immediately upstream of the translation start site direct expression of the b-glucuronidase reporter gene primarily in the vascular tissues, consistent with the phenotypes of the fbr6 mutant. AtSPL14 activates transcription in yeast, with a transactivation domain residing within the N-terminal region of the protein. Recombinant AtSPL14 protein binds A. thaliana genomic DNA in vitro in the absence of other proteins. These results indicate that FBR6/SPL14 functions as a transcriptional regulator that plays a role not only in sensitivity to FB1, but also in the development of normal plant architecture.
Proline metabolism has an underlying role in apoptotic signaling that impacts tumorigenesis. Proline is oxidized to glutamate in the mitochondria with the rate limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53 leading to increased proline oxidation, reactive oxygen species (ROS) formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and retained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress whereas knockdown of Δ1-pyrroline-5-carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the fork head transcription factor class O3a (FoxO3a). The role of PRODH in proline mediated protection was validated in the prostate carcinoma cell line, PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide mediated cell death and that proline/PRODH helps activate Akt in cancer cells.
The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.
Honey produced from medicinal plants hold great promise for human gut health.
SUMMARY During autophagy, phagophores grow into doublemembrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or nonvesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.
Electrocatalytic CO2 reduction reaction (CRR) is one of the most promising strategies to convert greenhouse gases to energy sources. Herein, the CRR was applied towards making C1 products (CO, HCOOH, CH3OH, and CH4) on g‐C3N4 frameworks with single Ni, Co, and Fe introduction; this process was investigated by density functional theory. The structures of the electrocatalysts, CO2 adsorption configurations, and CO2 reduction mechanisms were systematically studied. Results showed that the single Ni, Co, and Fe located from the corner of the g‐C3N4 cavity to the center. Analyses of the adsorption configurations and electronic structures suggested that CO2 could be chemically adsorbed on Co‐C3N4 and Fe‐C3N4, but physically adsorbed on Ni‐C3N4. The H2 evolution reaction (HER), as a suppression of CRR, was investigated, and results showed that Ni‐C3N4, Co‐C3N4, and Fe‐C3N4 exhibited more CRR selectivity than HER. CRR proceeded via COOH and OCHO as initial protonation intermediates on Ni‐C3N4 and Co/Fe‐C3N4, respectively, which resulted in different C1 products along quite different reaction pathways. Compared with Ni‐C3N4 and Fe‐C3N4, Co‐C3N4 had more favorable CRR activity and selectivity for CH3OH production with unique rate‐limiting steps and lower limiting potential.
Proteins with a conserved Cys- and His-rich SQUAMOSA promoter binding protein (SBP) domain are transcription factors restricted to photosynthetic organisms that possess a novel two Zn-finger structure DNA-binding domain. Despite the fact that altered expression of some SBP-encoding genes has profound effects on organism growth and development, little is known about SBP domain protein target genes. Misexpression of the Arabidopsis thaliana AtSPL14 SBP domain gene confers resistance to programmed cell death and modifies plant architecture. A consensus DNA-binding motif for AtSPL14 was identified by systematic evolution of ligands by exponential enrichment (SELEX) or random binding site selection (RBSS). DNA recognized by AtSPL14 contained the core binding motif (GTAC) found for other SBP domain proteins, but mutational analyses indicated that at least one additional flanking nucleotide is necessary for effective AtSPL14−DNA interaction. Comparison of several SBP domain amino acid sequences allows us to hypothesize which specific amino acids might participate in this sequence-specific DNA recognition. Electrophoretic mobility shift assays (EMSA) with mutant AtSPL14 DNA-binding domain proteins indicated that not all of the Zn2+ ion coordinating ligands in the second Zn structure are strictly required for DNA binding. Surface plasmon resonance (SPR) was used to evaluate AtSPL14 in vitro binding kinetics for comparison of equilibrium binding constants with other SBP domain proteins. These data provide a strong basis for further experiments aimed at defining and distinguishing the sets of genes regulated by the closely related SBP domain family members.
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