Abstract P239: Identification Of Binding Sites For Phosphodiesterase 5 (PDE5) On Rho-related BTB Domain Containing 1 (RhoBTB1) As A Guide To Identifying Novel RhoBTB1-Binding Partners

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Gaurav Kumar ◽  
Ko-Ting Lu ◽  
Wu Jing ◽  
Shi Fang ◽  
Curt D Sigmund
Keyword(s):  
Complex Disease ◽  
Isotope Labeling ◽  
Hek293 Cells ◽  
Proteomic Profiling ◽  
Aortic Smooth Muscle ◽  
Btb Domain ◽  
Intracellular Cgmp ◽  
Binding Interface ◽  
Ubiquitin Ligase Complex ◽  
Cullin 3

In-depth studies of molecular pathways have provided insights to explore therapeutic targets for treating hypertension as a complex disease. We previously identified PDE5 as an interacting partner with RhoBTB1 protein in aortic smooth muscle cells (SMCs). RhoBTB1 regulates PDE5 activity and therefore intracellular cGMP levels, and restoration of RhoBTB1 expression in RhoBTB1-deficient states results in improvement of cardiovascular status in a mouse model of hypertension. Here we identified the specific regions of RhoBTB1 that are responsible for the recruitment of PDE5. Our goal is to use this as a guide to identify additional RhoBTB1 interacting proteins which play important roles in cardiovascular function in the hope their identification may open novel therapeutic avenues against hypertension and associated pathologies. We hypothesize that RhoBTB1 acts as an adaptor for Cullin-3 E3 ring ubiquitin ligase complex, where Cullin-3 functions as “scaffold” which delivers PDE5 (and other targets) to Cullin-3 complex followed by ubiquitination and proteasomal degradation. We generated several truncations in full length RhoBTB1, splitting it into its GTPase, BTB1, BTB2, Carboxyl-terminal (CT), BTB1-BTB2, and BTB1-BTB2-CT domains. To check the preferred binding interface for PDE5, we co-transfected HEK293 cells with epitope tagged PDE5 and separately tagged truncations of RhoBTB1, and analyzed the interaction between PDE5 and RhoBTB1 domains using co-immunoprecipitation assays. Our result shows that BTB1-BTB2-C domain of RhoBTB1 is the preferred binding region for PDE5. The BTB1-BTB2 domain lacking the CT was unable to bind to PDE5. Next, we will utilize stable isotope labeling by amino acids in cell culture (SILAC) followed by proteomic profiling to identify additional targets and/or adaptors involved in the binding of PDE5 and RhoBTB1. Similarly, we will tag specific domains of RhoBTB1 using the ascorbate peroxidase (APEX2) fusion system followed by proteomic profiling.

scholarly journals Bioenergetic and Proteomic Profiling of Immune Cells in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients: An Exploratory Study

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 961
Author(s):  
Paula Fernandez-Guerra ◽  
Ana C. Gonzalez-Ebsen ◽  
Susanne E. Boonen ◽  
Julie Courraud ◽  
Niels Gregersen ◽  
...  
Keyword(s):  
Chronic Fatigue Syndrome ◽  
Complex Disease ◽  
Mononuclear Cells ◽  
Coupling Efficiency ◽  
Well Being ◽  
Chronic Fatigue ◽  
Myalgic Encephalomyelitis ◽  
Flux Analysis ◽  
Proteomic Profiling ◽  
Fatigue Syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous, debilitating, and complex disease. Along with disabling fatigue, ME/CFS presents an array of other core symptoms, including autonomic nervous system (ANS) dysfunction, sustained inflammation, altered energy metabolism, and mitochondrial dysfunction. Here, we evaluated patients' symptomatology and the mitochondrial metabolic parameters in peripheral blood mononuclear cells (PBMCs) and plasma from a clinically well-characterised cohort of six ME/CFS patients compared to age- and gender-matched controls. We performed a comprehensive cellular assessment using bioenergetics (extracellular flux analysis) and protein profiles (quantitative mass spectrometry-based proteomics) together with self-reported symptom measures of fatigue, ANS dysfunction, and overall physical and mental well-being. This ME/CFS cohort presented with severe fatigue, which correlated with the severity of ANS dysfunction and overall physical well-being. PBMCs from ME/CFS patients showed significantly lower mitochondrial coupling efficiency. They exhibited proteome alterations, including altered mitochondrial metabolism, centred on pyruvate dehydrogenase and coenzyme A metabolism, leading to a decreased capacity to provide adequate intracellular ATP levels. Overall, these results indicate that PBMCs from ME/CFS patients have a decreased ability to fulfill their cellular energy demands.

scholarly journals NRF2 and the Ambiguous Consequences of Its Activation during Initiation and the Subsequent Stages of Tumourigenesis

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3609
Author(s):  
Holly Robertson ◽  
Albena T. Dinkova-Kostova ◽  
John D. Hayes
Keyword(s):  
Somatic Mutations ◽  
Target Genes ◽  
Cancer Chemoprevention ◽  
Chemical Carcinogenesis ◽  
Related Factor ◽  
Gene Encoding ◽  
Ubiquitin Ligase Complex ◽  
Genes Encoding ◽  
Short Term Adaptation ◽  
Cullin 3

NF-E2 p45-related factor 2 (NRF2, encoded in the human by NFE2L2) mediates short-term adaptation to thiol-reactive stressors. In normal cells, activation of NRF2 by a thiol-reactive stressor helps prevent, for a limited period of time, the initiation of cancer by chemical carcinogens through induction of genes encoding drug-metabolising enzymes. However, in many tumour types, NRF2 is permanently upregulated. In such cases, its overexpressed target genes support the promotion and progression of cancer by suppressing oxidative stress, because they constitutively increase the capacity to scavenge reactive oxygen species (ROS), and they support cell proliferation by increasing ribonucleotide synthesis, serine biosynthesis and autophagy. Herein, we describe cancer chemoprevention and the discovery of the essential role played by NRF2 in orchestrating protection against chemical carcinogenesis. We similarly describe the discoveries of somatic mutations in NFE2L2 and the gene encoding the principal NRF2 repressor, Kelch-like ECH-associated protein 1 (KEAP1) along with that encoding a component of the E3 ubiquitin-ligase complex Cullin 3 (CUL3), which result in permanent activation of NRF2, and the recognition that such mutations occur frequently in many types of cancer. Notably, mutations in NFE2L2, KEAP1 and CUL3 that cause persistent upregulation of NRF2 often co-exist with mutations that activate KRAS and the PI3K-PKB/Akt pathway, suggesting NRF2 supports growth of tumours in which KRAS or PKB/Akt are hyperactive. Besides somatic mutations, NRF2 activation in human tumours can occur by other means, such as alternative splicing that results in a NRF2 protein which lacks the KEAP1-binding domain or overexpression of other KEAP1-binding partners that compete with NRF2. Lastly, as NRF2 upregulation is associated with resistance to cancer chemotherapy and radiotherapy, we describe strategies that might be employed to suppress growth and overcome drug resistance in tumours with overactive NRF2.

scholarly journals Revisiting the NaCl cotransporter regulation by with-no-lysine kinases

2015 ◽  
Vol 308 (10) ◽  
pp. C779-C791 ◽  
Author(s):  
Silvana Bazúa-Valenti ◽  
Gerardo Gamba
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Type Ii ◽  
Complex Issue ◽  
Dark Ages ◽  
Ubiquitin Ligase Complex ◽  
Pseudohypoaldosteronism Type Ii ◽  
Cullin 3 ◽  
Increased Activity ◽  
Pseudohypoaldosteronism Type

The renal thiazide-sensitive Na+-Cl− cotransporter (NCC) is the salt transporter in the distal convoluted tubule. Its activity is fundamental for defining blood pressure levels. Decreased NCC activity is associated with salt-remediable arterial hypotension with hypokalemia (Gitelman disease), while increased activity results in salt-sensitive arterial hypertension with hyperkalemia (pseudohypoaldosteronism type II; PHAII). The discovery of four different genes causing PHAII revealed a complex multiprotein system that regulates the activity of NCC. Two genes encode for with-no-lysine (K) kinases WNK1 and WNK4, while two encode for kelch-like 3 (KLHL3) and cullin 3 (CUL3) proteins that form a RING type E3 ubiquitin ligase complex. Extensive research has shown that WNK1 and WNK4 are the targets for the KLHL3-CUL3 complex and that WNKs modulate the activity of NCC by means of intermediary Ste20-type kinases known as SPAK or OSR1. The understanding of the effect of WNKs on NCC is a complex issue, but recent evidence discussed in this review suggests that we could be reaching the end of the dark ages regarding this matter.

scholarly journals The sleep gene insomniac ubiquitinates targets at postsynaptic densities and is required for retrograde homeostatic signaling

2018 ◽  
Author(s):  
Koto Kikuma ◽  
Xiling Li ◽  
Sarah Perry ◽  
Qiuling Li ◽  
Pragya Goel ◽  
...  
Keyword(s):  
Synaptic Strength ◽  
Homeostatic Plasticity ◽  
Retrograde Signaling ◽  
Sleep Regulation ◽  
Sleep Behavior ◽  
Receptor Inhibition ◽  
Induction Process ◽  
Ubiquitin Ligase Complex ◽  
Cullin 3 ◽  
Normal Sleep

ABSTRACTThe nervous system confronts challenges during development and experience that can destabilize information processing. To adapt to these perturbations, synapses homeostatically adjust synaptic strength, a process referred to as homeostatic synaptic plasticity. At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we designed a forward genetic screen to identify factors necessary in the postsynaptic compartment to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a gene that encodes a putative adaptor for the Cullin-3 ubiquitin ligase complex and is essential for normal sleep regulation. Intriguingly, we find that Inc rapidly traffics to postsynaptic densities and is required for increased ubiquitination following acute receptor inhibition. Our study suggests that Inc-dependent ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep behavior and homeostatic plasticity at synapses.

scholarly journals The E3 ligase adaptor molecule SPOP regulates fetal hemoglobin levels in adult erythroid cells

2019 ◽  
Vol 3 (10) ◽  
pp. 1586-1597 ◽  
Author(s):  
Xianjiang Lan ◽  
Eugene Khandros ◽  
Peng Huang ◽  
Scott A. Peslak ◽  
Saurabh K. Bhardwaj ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Messenger Rna ◽  
Fetal Hemoglobin ◽  
Dominant Negative ◽  
Protein Domain ◽  
Erythroid Cells ◽  
Protein Levels ◽  
Btb Domain ◽  
Chromatin Regulators ◽  
Ubiquitin Ligase Complex

Abstract Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and β-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain–focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain–containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of β-hemoglobinopathies.

scholarly journals The SPRY domain–containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation

2010 ◽  
Vol 190 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Zhihe Kuang ◽  
Rowena S. Lewis ◽  
Joan M. Curtis ◽  
Yifan Zhan ◽  
Bernadette M. Saunders ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Proteasomal Degradation ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Spry Domain ◽  
Persistent Infections ◽  
Binding Interface ◽  
Ubiquitin Ligase Complex

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain–containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB–iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.

scholarly journals Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

2012 ◽  
Vol 197 (1) ◽  
pp. 141-160 ◽  
Author(s):  
Georg H.H. Borner ◽  
Robin Antrobus ◽  
Jennifer Hirst ◽  
Gary S. Bhumbra ◽  
Patrycja Kozik ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Isotope Labeling ◽  
Principal Component ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Coat Proteins ◽  
Proteomic Profiling ◽  
Transport Vesicles ◽  
Associated Proteins ◽  
Sirna Knockdown

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.

Sign in / Sign up

Export Citation Format

Share Document