scholarly journals In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

2019 ◽  
Author(s):  
Alison Gillingham ◽  
Jessie Bertram ◽  
Farida Begum ◽  
Sean Munro
Keyword(s):  
Cell Growth ◽  
Membrane Traffic ◽  
Effector Proteins ◽  
Rab Gtpases ◽  
Wide Range ◽  
Ras Superfamily ◽  
Ras Family ◽  
Regulate Cell Growth

AbstractThe GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5 and Rab9. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.

scholarly journals In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alison K Gillingham ◽  
Jessie Bertram ◽  
Farida Begum ◽  
Sean Munro
Keyword(s):  
Cell Growth ◽  
Membrane Traffic ◽  
Effector Proteins ◽  
Rab Gtpases ◽  
Wide Range ◽  
Ras Superfamily ◽  
Ras Family ◽  
Regulate Cell Growth

The GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5, Rab9 and Rab11. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.

scholarly journals RASSF effectors couple diverse RAS subfamily GTPases to the Hippo pathway

2020 ◽  
Vol 13 (653) ◽  
pp. eabb4778 ◽  
Author(s):  
Thillaivillalan Dhanaraman ◽  
Swati Singh ◽  
Ryan C. Killoran ◽  
Anamika Singh ◽  
Xingjian Xu ◽  
...  
Keyword(s):  
Hippo Pathway ◽  
Effector Proteins ◽  
Domain Family ◽  
Ras Gtpases ◽  
Downstream Effector ◽  
Ras Superfamily ◽  
Ras Family ◽  
Key Residues ◽  
The Hippo Pathway ◽  
Apoptotic Induction

Small guanosine triphosphatases (GTPases) of the RAS superfamily signal by directly binding to multiple downstream effector proteins. Effectors are defined by a folded RAS-association (RA) domain that binds exclusively to GTP-loaded (activated) RAS, but the binding specificities of most RA domains toward more than 160 RAS superfamily GTPases have not been characterized. Ten RA domain family (RASSF) proteins comprise the largest group of related effectors and are proposed to couple RAS to the proapoptotic Hippo pathway. Here, we showed that RASSF1-6 formed complexes with the Hippo kinase ortholog MST1, whereas RASSF7-10 formed oligomers with the p53-regulating effectors ASPP1 and ASPP2. Moreover, only RASSF5 bound directly to activated HRAS and KRAS, and RASSFs did not augment apoptotic induction downstream of RAS oncoproteins. Structural modeling revealed that expansion of the RASSF effector family in vertebrates included amino acid substitutions to key residues that direct GTPase-binding specificity. We demonstrated that the tumor suppressor RASSF1A formed complexes with the RAS-related GTPases GEM, REM1, REM2, and the enigmatic RASL12. Furthermore, interactions between RASSFs and RAS GTPases blocked YAP1 nuclear localization. Thus, these simple scaffolds link the activation of diverse RAS family small G proteins to Hippo or p53 regulation.

scholarly journals An Emerging Role for IQGAP1 in Regulating Protein Traffic

2010 ◽  
Vol 10 ◽  
pp. 944-953 ◽  
Author(s):  
Mahasin Osman
Keyword(s):  
Cell Growth ◽  
Specific Inhibitor ◽  
Membrane Traffic ◽  
Mtor Pathway ◽  
Human Diseases ◽  
Multifunctional Protein ◽  
Intracellular Traffic ◽  
Human Cancers ◽  
C Terminus ◽  
Wide Range

IQGAP1, an effector of CDC42p GTPase, is a widely conserved, multifunctional protein that bundles F-actin through its N-terminus and binds microtubules through its C-terminus to modulate the cell architecture. It has emerged as a potential oncogene associated with diverse human cancers. Therefore, IQGAP1 has been heavily investigated; regardless, its precise cellular function remains unclear. Work from yeast suggests that IQGAP1 plays an important role in directed cell growth, which is a conserved feature crucial to morphogenesis, division axis, and body plan determination. New evidence suggests a conserved role for IQGAP1 in protein synthesis and membrane traffic, which may help to explain the diversity of its cellular functions. Membrane traffic mediates infections by intracellular pathogens and a range of degenerative human diseases arise from dysfunctions in intracellular traffic; thus, elucidating the mechanisms of cellular traffic will be important in order to understand the basis of a wide range of inherited and acquired human diseases. Recent evidence suggests that IQGAP1 plays its role in cell growth through regulating the conserved mTOR pathway. The mTOR signaling cascade has been implicated in membrane traffic and is activated in nearly all human cancers, but clinical response to the mTOR-specific inhibitor rapamycin has been disappointing. Thus, understanding the regulators of this pathway will be crucial in order to identify predictors of rapamycin sensitivity. In this review, I discuss emerging evidence that supports a potential role of IQGAP1 in regulating membrane traffic via regulating the mTOR pathway.

scholarly journals Role of Rab GTPases in Membrane Traffic and Cell Physiology

2011 ◽  
Vol 91 (1) ◽  
pp. 119-149 ◽  
Author(s):  
Alex H. Hutagalung ◽  
Peter J. Novick
Keyword(s):  
Membrane Traffic ◽  
Effector Proteins ◽  
Cell Physiology ◽  
Rab Gtpases ◽  
Disease States ◽  
Transport Vesicle ◽  
Target Membrane ◽  
The Many ◽  
Vesicle Movement ◽  
Regulatory Functions

Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.

scholarly journals A monoclonal antibody against CD148, a receptor-like tyrosine phosphatase, inhibits endothelial-cell growth and angiogenesis

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1234-1242 ◽  
Author(s):  
Takamune Takahashi ◽  
Keiko Takahashi ◽  
Raymond L. Mernaugh ◽  
Nobuo Tsuboi ◽  
Hua Liu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Cell Cycle Progression ◽  
Vascular Endothelial Cells ◽  
Tyrosine Phosphatase ◽  
Vessel Formation ◽  
Wide Range ◽  
Endothelial Cell Growth

Abstract Angiogenesis contributes to a wide range of neoplastic, ischemic, and inflammatory disorders. Definition of the intrinsic molecular controls in angiogenic vessel growth promises novel therapeutic approaches for angiogenesis-related diseases. CD148 (also named DEP-1/PTPη) is a receptor-like protein tyrosine phosphatase that is abundantly expressed in vascular endothelial cells. To explore a role of CD148 in endothelial vessel formation, we generated a monoclonal antibody, Ab1, against the ectodomain sequence of CD148 and examined its effects on endothelial-cell growth and vessel formation. Here we report that a bivalent, but not a monovalent, form of the Ab1 antibody inhibits endothelial-cell growth and blocks angiogenesis in mouse cornea in vivo. We further demonstrate that (1) bivalent Ab1 arrests cell-cycle progression of CD148-transfected CHO cells at G0/G1 phase, (2) coexpression of catalytically inactive CD148 mutants attenuates the Ab1-cell growth inhibition, and (3) bivalent Ab1 suppresses phosphorylation of ERK1/2 kinases and Met tyrosine kinase as activated CD148 does, with an increase in CD148-associated tyrosine phosphatase activity. Taken together, these findings demonstrate that Ab1-induced ectodomain oligomerization arrests endothelial-cell growth through catalytic activity of the CD148 cytoplasmic domain. The present study defines CD148 as a valuable molecular target for antiangiogenesis therapy.

scholarly journals Abl Interactor 1 Binds to Sos and Inhibits Epidermal Growth Factor- and v-Abl-Induced Activation of Extracellular Signal-Regulated Kinases

2000 ◽  
Vol 20 (20) ◽  
pp. 7591-7601 ◽  
Author(s):  
Pang-Dian Fan ◽  
Stephen P. Goff
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Cell Growth ◽  
Epidermal Growth Factor ◽  
Nucleotide Exchange ◽  
Amino Terminal ◽  
Extracellular Signal ◽  
Regulate Cell Growth ◽  
Epidermal Growth

ABSTRACT Recent studies have suggested that members of the Abl interactor (Abi) protein family negatively regulate cell growth and transformation. To date, however, no specific role in these cellular processes has been identified for the Abi family. Here we describe the inhibition by overexpressed Abi-1 of a mitogenic pathway activated by both growth factors and v-Abl. We have identified the guanine nucleotide exchange factors Sos1 and Sos2 as novel binding partners of Abi-1. A domain that is required for interaction with Sos in vivo has been mapped to the amino terminus of Abi-1. Overexpression of Abi-1 inhibits epidermal growth factor (EGF)-induced activation of extracellular signal-regulated kinases (Erks) but does not affect EGF-induced activation of c-Jun N-terminal kinase or Akt. In addition, overexpression of Abi-1 blocks Erk activation induced by v-Abl. In both cases, the maximal inhibitory effect requires an intact amino-terminal Sos-binding domain in Abi-1. Finally, we demonstrate that tyrosine phosphorylation of endogenous Abi-1 in fibroblasts is induced by both v-Abl and serum stimulation, further suggesting a role for Abi-1 in signal transduction initiated by v-Abl and growth factors. Taken together, these findings suggest that overexpressed Abi proteins negatively regulate cell growth and transformation by specifically targeting the Erk pathway.

scholarly journals Bnip3 Mediates the Hypoxia-induced Inhibition on Mammalian Target of Rapamycin by Interacting with Rheb

2007 ◽  
Vol 282 (49) ◽  
pp. 35803-35813 ◽  
Author(s):  
Yong Li ◽  
Yian Wang ◽  
Eunjung Kim ◽  
Peter Beemiller ◽  
Cun-Yu Wang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Energy Levels ◽  
Mammalian Target Of Rapamycin ◽  
Small Gtpase ◽  
Mtor Pathway ◽  
Target Of Rapamycin ◽  
Intracellular Signals ◽  
Central Controller ◽  
Wide Range

The mammalian target of rapamycin (mTOR) is a central controller of cell growth, and it regulates translation, cell size, cell viability, and cell morphology. mTOR integrates a wide range of extracellular and intracellular signals, including growth factors, nutrients, energy levels, and stress conditions. Rheb, a Ras-related small GTPase, is a key upstream activator of mTOR. In this study, we found that Bnip3, a hypoxia-inducible Bcl-2 homology 3 domain-containing protein, directly binds Rheb and inhibits the mTOR pathway. Bnip3 decreases Rheb GTP levels in a manner depending on the binding to Rheb and the presence of the N-terminal domain. Both knockdown and overexpression experiments show that Bnip3 plays an important role in mTOR inactivation in response to hypoxia. Moreover, Bnip3 inhibits cell growth in vivo by suppressing the mTOR pathway. These observations demonstrate that Bnip3 mediates the inhibition of the mTOR pathway in response to hypoxia.

scholarly journals Stress induces the transcription of toxin-antitoxin systems but does not activate toxin

2020 ◽  
Author(s):  
Michele LeRoux ◽  
Peter H. Culviner ◽  
Yue J. Liu ◽  
Megan L. Littlehale ◽  
Michael T. Laub
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Sequencing ◽  
Bacterial Genomes ◽  
Transcript Levels ◽  
Mrna Cleavage ◽  
Regulate Cell Growth ◽  
Response To Stress ◽  
Reliable Marker

AbstractToxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking 10 TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA-sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems likely do not strongly promote survival following stress.

scholarly journals Counteracting the Ramifications of UVB Irradiation and Photoaging with Swietenia macrophylla King Seed

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 2000
Author(s):  
Camille Keisha Mahendra ◽  
Syafiq Asnawi Zainal Abidin ◽  
Thet Thet Htar ◽  
Lay-Hong Chuah ◽  
Shafi Ullah Khan ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Growth ◽  
Redox System ◽  
Ethyl Acetate Fraction ◽  
Collagen Degradation ◽  
Swietenia Macrophylla ◽  
Molecular Changes ◽  
Uvb Irradiation ◽  
Wide Range

In this day and age, the expectation of cosmetic products to effectively slow down skin photoaging is constantly increasing. However, the detrimental effects of UVB on the skin are not easy to tackle as UVB dysregulates a wide range of molecular changes on the cellular level. In our research, irradiated keratinocyte cells not only experienced a compromise in their redox system, but processes from RNA translation to protein synthesis and folding were also affected. Aside from this, proteins involved in various other processes like DNA repair and maintenance, glycolysis, cell growth, proliferation, and migration were affected while the cells approached imminent cell death. Additionally, the collagen degradation pathway was also activated by UVB irradiation through the upregulation of inflammatory and collagen degrading markers. Nevertheless, with the treatment of Swietenia macrophylla (S. macrophylla) seed extract and fractions, the dysregulation of many genes and proteins by UVB was reversed. The reversal effects were particularly promising with the S. macrophylla hexane fraction (SMHF) and S. macrophylla ethyl acetate fraction (SMEAF). SMHF was able to oppose the detrimental effects of UVB in several different processes such as the redox system, DNA repair and maintenance, RNA transcription to translation, protein maintenance and synthesis, cell growth, migration and proliferation, and cell glycolysis, while SMEAF successfully suppressed markers related to skin inflammation, collagen degradation, and cell apoptosis. Thus, in summary, our research not only provided a deeper insight into the molecular changes within irradiated keratinocytes, but also serves as a model platform for future cosmetic research to build upon. Subsequently, both SMHF and SMEAF also displayed potential photoprotective properties that warrant further fractionation and in vivo clinical trials to investigate and obtain potential novel bioactive compounds against photoaging.

scholarly journals Evaluation of Annona muricata Acetogenins as Potential Anti-SARS-CoV-2 Agents Through Computational Approaches

2021 ◽  
Vol 8 ◽  
Author(s):  
Shashanka K. Prasad ◽  
Sushma Pradeep ◽  
Chandan Shimavallu ◽  
Shiva Prasad Kollur ◽  
Asad Syed ◽  
...  
Keyword(s):  
Cell Growth ◽  
Bioactive Compounds ◽  
In Silico ◽  
Spike Protein ◽  
Annona Muricata ◽  
Annonaceous Acetogenins ◽  
Tropical Plant ◽  
Wide Range

Annona muricata, a tropical plant which has been extensively used in ethnomedicine to treat a wide range of diseases, from malaria to cancer. Interestingly, this plant has been reported to demonstrate significant antiviral properties against the human immunodeficiency virus, herpes simplex virus, human papilloma virus, hepatitis C virus and dengue virus. Additionally, the bioactive compounds responsible for antiviral efficacy have also shown to be selectively cytotoxic while inhibiting tumorigenic cell growth without affecting the normal cell growth. Annonaceous Acetogenins are a class of bioactive compounds exclusive to the Annonaceae family at which the plant A. muricata belongs. In the current study, we have created a library of Acetogenins unique to the plant, comprising of Annomuricin A, Annomuricin B, Annomuricin C, Muricatocin C, Muricatacin, cis-Annonacin, Annonacin-10-one, cis-Goniothalamicin, Arianacin and Javoricin, for in silico and theoretical evaluations against the SARS-CoV-2 spike protein in an attempt toward promotion of plant based drug development for the current pandemic of coronavirus disease 2019 (COVID-19). We found that all the Acetogenins showing in silico spike protein significantly docking with good binding affinities. Moreover, we envision A. muricata Acetogenins can be further studied by in vitro and in vivo models to identify potential anti-SARS-CoV-2 agents.

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