scholarly journals The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila

2020 ◽  
Vol 117 (4) ◽  
pp. 2004-2013 ◽  
Author(s):  
Kenneth Kin Lam Wong ◽  
Ta-Wei Liu ◽  
Jessica M. Parker ◽  
Donald A. R. Sinclair ◽  
Yi-Yun Chen ◽  
...  
Keyword(s):  
Protein Stability ◽  
Mammalian Cells ◽  
Normal Diet ◽  
Cancer Proliferation ◽  
Hexosamine Biosynthetic Pathway ◽  
Interacting Protein ◽  
Molecular Sensors ◽  
Cellular Behaviors ◽  
Glcnac Transferase ◽  
Necessary And Sufficient

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.

Early signalling events of autophagy

2013 ◽  
Vol 55 ◽  
pp. 1-15 ◽  
Author(s):  
Laura E. Gallagher ◽  
Edmond Y.W. Chan
Keyword(s):  
Protein Kinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Interacting Protein ◽  
The Core ◽  
Autophagy Gene ◽  
Autophagy Induction ◽  
Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

scholarly journals MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

2017 ◽  
Vol 114 (36) ◽  
pp. E7450-E7459 ◽  
Author(s):  
Shuzhen Liu ◽  
Hua Liu ◽  
Andrea Johnston ◽  
Sarah Hanna-Addams ◽  
Eduardo Reynoso ◽  
...  
Keyword(s):  
Cell Death ◽  
Disulfide Bond ◽  
Kinase Domain ◽  
Proteinase K ◽  
Interacting Protein ◽  
Tnf Α ◽  
Mouse Cells ◽  
Red Dye ◽  
Necessary And Sufficient ◽  
Human And Mouse

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α–induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

scholarly journals NudCL2 regulates cell migration by stabilizing both myosin-9 and LIS1 with Hsp90

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Wenwen Chen ◽  
Wei Wang ◽  
Xiaoxia Sun ◽  
Shanshan Xie ◽  
Xiaoyang Xu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Single Cell ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Heat Shock Protein 90 ◽  
Underlying Mechanism ◽  
Collective Cell Migration ◽  
Interacting Protein ◽  
Protein Levels ◽  
Protein Instability

Abstract Cell migration plays pivotal roles in many biological processes; however, its underlying mechanism remains unclear. Here, we find that NudC-like protein 2 (NudCL2), a cochaperone of heat shock protein 90 (Hsp90), modulates cell migration by stabilizing both myosin-9 and lissencephaly protein 1 (LIS1). Either knockdown or knockout of NudCL2 significantly increases single-cell migration, but has no significant effect on collective cell migration. Immunoprecipitation–mass spectrometry and western blotting analyses reveal that NudCL2 binds to myosin-9 in mammalian cells. Depletion of NudCL2 not only decreases myosin-9 protein levels, but also results in actin disorganization. Ectopic expression of myosin-9 efficiently reverses defects in actin disorganization and single-cell migration in cells depleted of NudCL2. Interestingly, knockdown of myosin-9 increases both single and collective cell migration. Depletion of LIS1, a NudCL2 client protein, suppresses both single and collective cell migration, which exhibits the opposite effect compared with myosin-9 depletion. Co-depletion of myosin-9 and LIS1 promotes single-cell migration, resembling the phenotype caused by NudCL2 depletion. Furthermore, inhibition of Hsp90 ATPase activity also reduces the Hsp90-interacting protein myosin-9 stability and increases single-cell migration. Forced expression of Hsp90 efficiently reverses myosin-9 protein instability and the defects induced by NudCL2 depletion, but not vice versa. Taken together, these data suggest that NudCL2 plays an important role in the precise regulation of cell migration by stabilizing both myosin-9 and LIS1 via Hsp90 pathway.

scholarly journals O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yunfan Yang ◽  
Minnie Fu ◽  
Min-Dian Li ◽  
Kaisi Zhang ◽  
Bichen Zhang ◽  
...  
Keyword(s):  
Visceral Fat ◽  
Hexosamine Biosynthetic Pathway ◽  
Visceral Fat Accumulation ◽  
Diet Induced Obesity ◽  
Treatment Of Obesity ◽  
High Level ◽  
Glcnac Transferase ◽  
Metabolically Unhealthy Obesity ◽  
Unique Potential ◽  
Perilipin 1

AbstractExcessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.

scholarly journals Homozygous mutations in DZIP1 can induce asthenoteratospermia with severe MMAF

2020 ◽  
Vol 57 (7) ◽  
pp. 445-453 ◽  
Author(s):  
Mingrong Lv ◽  
Wangjie Liu ◽  
Wangfei Chi ◽  
Xiaoqing Ni ◽  
Jiajia Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Clinical Manifestations ◽  
Interacting Protein ◽  
Genome Data ◽  
Pathogenic Genes ◽  
Whole Exome ◽  
Exome Aggregation Consortium ◽  
Common Causes ◽  
Deleted In Azoospermia ◽  
The Common

BackgroundAsthenoteratospermia, one of the most common causes for male infertility, often presents with defective sperm heads and/or flagella. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the common clinical manifestations of asthenoteratospermia. Variants in several genes including DNAH1, CEP135, CATSPER2 and SUN5 are involved in the genetic pathogenesis of asthenoteratospermia. However, more than half of the asthenoteratospermia cases cannot be explained by the known pathogenic genes.Methods and resultsTwo asthenoteratospermia-affected men with severe MMAF (absent flagella in >90% spermatozoa) from consanguineous families were subjected to whole-exome sequencing. The first proband had a homozygous missense mutation c.188G>A (p.Arg63Gln) of DZIP1 and the second proband had a homozygous stop-gain mutation c.690T>G (p.Tyr230*). Both of the mutations were neither detected in the human population genome data (1000 Genomes Project, Exome Aggregation Consortium) nor in our own data of a cohort of 875 Han Chinese control populations. DZIP1 encodes a DAZ (a protein deleted in azoospermia) interacting protein, which was associated with centrosomes in mammalian cells. Immunofluorescence staining of the centriolar protein Centrin1 indicated that the spermatozoa of the proband presented with abnormal centrosomes, including no concentrated centriolar dot or more than two centriolar dots. HEK293T cells transfected with two DZIP1-mutated constructs showed reduced DZIP1 level or truncated DZIP1. The Dzip1-knockout mice, generated by the CRSIPR-Cas9, revealed consistent phenotypes of severe MMAF.ConclusionOur study strongly suggests that homozygous DZIP1 mutations can induce asthenoteratospermia with severe MMAF. The deficiency of DZIP1 induces sperm centrioles dysfunction and causes the absence of flagella.

Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death

2010 ◽  
Vol 427 (1) ◽  
pp. 91-104 ◽  
Author(s):  
Yatender Kumar ◽  
Vegesna Radha ◽  
Ghanshyam Swarup
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Intramolecular Interaction ◽  
Dominant Negative ◽  
26S Proteasome ◽  
Caspase 8 ◽  
Interacting Protein ◽  
Caspase 1 ◽  
Lung Carcinoma Cells ◽  
Lrr Domain

Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1β-converting enzyme)-protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)-containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase 1. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91–253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-α (tumour necrosis factor α) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.

scholarly journals Knockdown of p180 Eliminates the Terminal Differentiation of a Secretory Cell Line

2009 ◽  
Vol 20 (2) ◽  
pp. 732-744 ◽  
Author(s):  
Payam Benyamini ◽  
Paul Webster ◽  
David I. Meyer
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Secretory Cell ◽  
Terminal Differentiation ◽  
Secretory Cells ◽  
Rnai Technology ◽  
Apo E ◽  
Secretory Phenotype ◽  
Rough Er ◽  
Necessary And Sufficient

We have previously reported that the expression in yeast of an integral membrane protein (p180) of the endoplasmic reticulum (ER), isolated for its ability to mediate ribosome binding, is capable of inducing new membrane biogenesis and an increase in secretory capacity. To demonstrate that p180 is necessary and sufficient for terminal differentiation and acquisition of a secretory phenotype in mammalian cells, we studied the differentiation of a secretory cell line where p180 levels had been significantly reduced using RNAi technology and by transiently expressing p180 in nonsecretory cells. A human monocytic (THP-1) cell line, that can acquire macrophage-like properties, failed to proliferate rough ER when p180 levels were lowered. The Golgi compartment and the secretion of apolipoprotein E (Apo E) were dramatically affected in cells expressing reduced p180 levels. On the other hand, expression of p180 in a human embryonic kidney nonsecretory cell line (HEK293) showed a significant increase in proliferation of rough ER membranes and Golgi complexes. The results obtained from knockdown and overexpression experiments demonstrate that p180 is both necessary and sufficient to induce a secretory phenotype in mammalian cells. These findings support a central role for p180 in the terminal differentiation of secretory cells and tissues.

scholarly journals Regulating Protein Stability in Mammalian Cells Using Small Molecules

2009 ◽  
Vol 2009 (3) ◽  
pp. pdb.prot5172-pdb.prot5172 ◽  
Author(s):  
E. L. Hagan ◽  
L. A. Banaszynski ◽  
L.-c. Chen ◽  
L. A. Maynard-Smith ◽  
T. J. Wandless
Keyword(s):  
Protein Stability ◽  
Small Molecules ◽  
Mammalian Cells

253. Toxic effects of hyperglycaemia arise from induced O-linked glycosylation in early mouse embryos

2008 ◽  
Vol 20 (9) ◽  
pp. 53
Author(s):  
M. Pantaleon ◽  
H. Tan ◽  
P. L. Kaye
Keyword(s):  
Cell Proliferation ◽  
Negative Impact ◽  
Cell Number ◽  
Signalling Pathway ◽  
Blastocyst Formation ◽  
Hexosamine Biosynthetic Pathway ◽  
Sense And Respond ◽  
Pathway Inhibition ◽  
Early Mouse ◽  
Glcnac Transferase

Glucose flux through the hexosamine biosynthetic pathway (HBP) which is essential for preimplantation development (1) produces uridine 5′-diphospho-N-acetylglucosamine, a donor substrate for multiple glycosylation reactions including O-linked glycosylation. This novel signalling arm of the HBP, known as the hexosamine signalling pathway (HSP) operates via reversible addition of an O-linked β-N-acetylglucosamine (O-GlcNAc) unit to serine and threonine residues of proteins including transcription factors, cytoskeletal components, metabolic enzymes and cellular signalling components. O-linked glycosylation is functionally reciprocal to phosphorylation at the same residues, altering the activity and/or stability of targeted proteins, thus providing a mechanism for modulating cellular physiology in response to glucose availability. The enzymes regulating this O-GlcNAcylation are the β-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective β-N-acetylglucosaminidase (O-GlcNAcase). We hypothesised that the toxicity of hyperglycemia on early embryos arises from increased flux through HBP and increased O-GlcNAcylation of key proteins. Mouse zygotes (18 h post hCG) were cultured under conditions of modified flux through the HSP including hypoglycemia, hyperglycemia or supplemented with glucosamine which feeds exclusively into the HBP to increase downstream O-GlcNAcylation. BADGP was used to inhibit OGT and O-GlcNAcylation. Blastocyst formation, cell proliferation and apoptosis were assessed. Treatments that perturb levels of intracellular protein O-GlcNAcylation inhibited embryo development. Whilst some flux through HBP is required to activate embryonic differentiation (1), excess flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this novel post-translational signalling pathway. Inhibition of OGT using 2 mM BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number and apoptosis supporting our hypothesis that O-GlcNAcylation is a key mechanism used by the embryo to sense and respond to perturbations of glucose in its environment. (1) Pantaleon M, Scott J and Kaye PL (2008) Biol Reprod, 78(4):595–600

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