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ACS Synthetic Biology Apr 2023peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed...
peptides and proteins that switch state in response to chemical and physical cues would advance protein design and synthetic biology. Here we report two designed systems that disassemble and reassemble upon site-specific phosphorylation and dephosphorylation, respectively. As starting points, we use hyperthermostable antiparallel and parallel coiled-coil heterotetramers, , AB systems, to afford control in downstream applications. The switches are incorporated by adding protein kinase A phosphorylation sites, R-R-X-S, with the phosphoacceptor serine residues placed to maximize disruption of the coiled-coil interfaces. The unphosphorylated peptides assemble as designed and unfold reversibly when heated. Addition of kinase to the assembled states unfolds them with half-lives of ≤5 min. Phosphorylation is reversed by Lambda Protein Phosphatase resulting in tetramer reassembly. We envisage that the new designed coiled-coil components, the switches, and a mechanistic model for them will be useful in synthetic biology, biomaterials, and biotechnology applications.
Topics: Phosphorylation; Protein Structure, Secondary; Peptides; Proteins; Protein Domains
PubMed: 36988263
DOI: 10.1021/acssynbio.3c00064 -
Cells Apr 2022Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in... (Review)
Review
Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A-D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation, -GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy.
Topics: Animals; Autophagy; Autophagy-Related Protein 8 Family; Autophagy-Related Proteins; Mammals; Microtubule-Associated Proteins; Peptide Hydrolases; Protein Processing, Post-Translational
PubMed: 35456009
DOI: 10.3390/cells11081330 -
Biomolecules Nov 2022Protein phosphorylation and dephosphorylation are widely considered to be the key regulatory factors of cell function, and are often referred to as "molecular switches"... (Review)
Review
Protein phosphorylation and dephosphorylation are widely considered to be the key regulatory factors of cell function, and are often referred to as "molecular switches" in the regulation of cell metabolic processes. A large number of studies have shown that the phosphorylation/dephosphorylation of related signal molecules plays a key role in the regulation of liver glucose and lipid metabolism. As a new therapeutic strategy for metabolic diseases, the potential of using inhibitor-based therapies to fight diabetes has gained scientific momentum. PTG, a protein phosphatase, also known as glycogen targeting protein, is a member of the protein phosphatase 1 (PP1) family. It can play a role by catalyzing the dephosphorylation of phosphorylated protein molecules, especially regulating many aspects of glucose and lipid metabolism. In this review, we briefly summarize the role of PTG in glucose and lipid metabolism, and update its role in metabolic regulation, with special attention to glucose homeostasis and lipid metabolism.
Topics: Glucose; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Protein Phosphatase 1; Glycogen
PubMed: 36551183
DOI: 10.3390/biom12121755 -
Trends in Plant Science Sep 2023The Salt Overly Sensitive (SOS) pathway plays a central role in plant salinity tolerance. Since the discovery of the SOS pathway, transcriptional and post-translational... (Review)
Review
The Salt Overly Sensitive (SOS) pathway plays a central role in plant salinity tolerance. Since the discovery of the SOS pathway, transcriptional and post-translational regulations of its core components have garnered considerable attention. To date, several proteins that regulate these core components, either positively or negatively at the protein and transcript levels, have been identified. Here, we review recent advances in the understanding of the functional regulation of the core proteins of the SOS pathway and an expanding spectrum of their upstream effectors in plants. Furthermore, we also discuss how these novel regulators act as key signaling nodes of multilayer control of plant development and stress adaptation through modulation of the SOS core proteins at the transcriptional and post-translational levels.
Topics: Salt Tolerance; Arabidopsis Proteins; Plant Proteins; Adaptation, Physiological; Gene Expression Regulation, Plant
PubMed: 37117077
DOI: 10.1016/j.tplants.2023.04.003 -
Nature Communications Apr 2023The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the...
The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. Our cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate phosphorylation sites neighboring the kinase domain. Thus, we directly show how Hsp90's role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.
Topics: Humans; Cell Cycle Proteins; Protein Binding; HSP90 Heat-Shock Proteins; Molecular Chaperones
PubMed: 37069154
DOI: 10.1038/s41467-023-37659-7 -
Experimental Biology and Medicine... Mar 2022PPM1A (magnesium-dependent phosphatase 1 A, also known as PP2Cα) is a member of the Ser/Thr protein phosphatase family. Protein phosphatases catalyze the removal of... (Review)
Review
PPM1A (magnesium-dependent phosphatase 1 A, also known as PP2Cα) is a member of the Ser/Thr protein phosphatase family. Protein phosphatases catalyze the removal of phosphate groups from proteins via hydrolysis, thus opposing the role of protein kinases. The PP2C family is generally considered a negative regulator in the eukaryotic stress response pathway. PPM1A can bind and dephosphorylate various proteins and is therefore involved in the regulation of a wide range of physiological processes. It plays a crucial role in transcriptional regulation, cell proliferation, and apoptosis and has been suggested to be closely related to the occurrence and development of cancers of the lung, bladder, and breast, amongst others. Moreover, it is closely related to certain autoimmune diseases and neurodegenerative diseases. In this review, we provide an insight into currently available knowledge of PPM1A, including its structure, biological function, involvement in signaling pathways, and association with diseases. Lastly, we discuss whether PPM1A could be targeted for therapy of certain human conditions.
Topics: Apoptosis; Gene Expression Regulation; Humans; Protein Phosphatase 2C; Signal Transduction
PubMed: 34861123
DOI: 10.1177/15353702211061883 -
Molecular & Cellular Proteomics : MCP Aug 2023Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to...
Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation sites and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent dephosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation.
Topics: Humans; Proteolysis; Protein Serine-Threonine Kinases; Phosphoprotein Phosphatases; Phosphorylation; Threonine; Colorectal Neoplasms; Protein Phosphatase 2
PubMed: 37392812
DOI: 10.1016/j.mcpro.2023.100614 -
Journal of Neurochemistry Dec 2022Subarachnoid haemorrhage (SAH) has a high rate of disability and mortality. Extremely damaging molecules, including adenosine triphosphate (ATP), are released from...
Subarachnoid haemorrhage (SAH) has a high rate of disability and mortality. Extremely damaging molecules, including adenosine triphosphate (ATP), are released from extravasated red blood cells and nerve cells, which activate microglia and induce sterile tissue injury and organ dysfunction. P2X purinoceptor 7 (P2X7) is one of the most important purine receptors on the microglial surface and is involved in the proinflammatory activation of microglia. While P2X7 can also affect microglial phagocytosis, the mechanism is not clear. Here, we demonstrated that microglial phagocytosis is progressively impaired under continued BzATP exposure and P2X7 activation. Furthermore, we found that P2X7 activation leads to increased intracellular Ca levels and activates Calcineurin, which dephosphorylates dynamin-related protein 1 (DRP1) S637. The dephosphorylation of DRP1 at S637 leads to increased mitochondrial fission and decreased mitochondrial function, which may be responsible for the decreased microglial phagocytosis. Finally, we pharmacologically inhibited P2X7 activation in mice, which resulted in rescue of mitochondrial function and decreased microglial proliferation, but improved phagocytosis after SAH. Our study confirmed that P2X7 activation after SAH leads to the impairment of microglial phagocytosis through mitochondrial fission and verified that P2X7 inhibition restores microglial phagocytosis both in vitro and in vivo.
Topics: Animals; Mice; Adenosine Triphosphate; Microglia; Mitochondria; Mitochondrial Dynamics; Phagocytosis; Receptors, Purinergic P2X7; Subarachnoid Hemorrhage; Humans
PubMed: 36269673
DOI: 10.1111/jnc.15712 -
Nature Communications Dec 2021Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit...
Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit its kinase activity and cell death. How these inhibitory phosphorylation sites are dephosphorylated is poorly understood. Using a sensitized CRISPR whole-genome knockout screen, we discover that protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is required for RIPK1-dependent apoptosis and type I necroptosis. Mechanistically, PPP1R3G recruits its catalytic subunit protein phosphatase 1 gamma (PP1γ) to complex I to remove inhibitory phosphorylations of RIPK1. A PPP1R3G mutant which does not bind PP1γ fails to rescue RIPK1 activation and cell death. Furthermore, chemical prevention of RIPK1 inhibitory phosphorylations or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, Ppp1r3g mice are protected from tumor necrosis factor-induced systemic inflammatory response syndrome, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo.
Topics: Animals; Apoptosis; Cell Line, Tumor; Disease Models, Animal; Humans; Mice; Mice, Knockout; Mutation; Necroptosis; Phosphorylation; Protein Phosphatase 1; Receptor-Interacting Protein Serine-Threonine Kinases; Systemic Inflammatory Response Syndrome; Tumor Necrosis Factor-alpha
PubMed: 34862394
DOI: 10.1038/s41467-021-27367-5 -
International Journal of Molecular... Jul 2023Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C),... (Review)
Review
Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'β clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'β being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.
Topics: Arabidopsis; Arabidopsis Proteins; Phosphorylation; Physiological Phenomena; Protein Phosphatase 2; Protein Subunits; Transcription Factors
PubMed: 37569631
DOI: 10.3390/ijms241512255