scholarly journals Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)

2021 ◽  
Vol 118 (12) ◽  
pp. e2023245118
Author(s):  
Nikolaus Dietz ◽  
Markus Huber ◽  
Isabel Sorg ◽  
Arnaud Goepfert ◽  
Alexander Harms ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Bacterial Toxins ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Structural Basis ◽  
Effector Protein ◽  
Rho Family ◽  
Specific Pair

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

scholarly journals Structural basis for selective targeting of Rac subfamily GTPases by a bacterial effector protein

2020 ◽  
Author(s):  
Nikolaus Balthasar Dietz ◽  
Markus Huber ◽  
Isabel Sorg ◽  
Arnaud Goepfert ◽  
Alexander Harms ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Molecular Mechanisms ◽  
Effector Proteins ◽  
Structural Basis ◽  
Effector Protein ◽  
Binding Motif ◽  
Selective Targeting ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Specific Pair

SummaryRas-homology (Rho) family GTPases are conserved molecular switches controlling fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, molecular mechanisms of target selectivity have remained elusive. Here, we report a bacterial effector protein that targets all four Rac subfamily members of Rho family GTPases, but none of the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 facilitate modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural paradigm for target selectivity towards Rac subfamily GTPases and provides a highly selective tool for their functional analysis.

Rho family GTPases

2012 ◽  
Vol 40 (6) ◽  
pp. 1378-1382 ◽  
Author(s):  
Alan Hall
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Cellular Response ◽  
Molecular Switches ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Cell Behaviour ◽  
Wide Range ◽  
Rho Family Gtpases ◽  
Rho Family

Rho GTPases comprise a family of molecular switches that control signal transduction pathways in eukaryotic cells. A conformational change induced upon binding GTP promotes an interaction with target (effector) proteins to generate a cellular response. A highly conserved function of Rho GTPases from yeast to humans is to control the actin cytoskeleton, although, in addition, they promote a wide range of other cellular activities. Changes in the actin cytoskeleton drive many dynamic aspects of cell behaviour, including morphogenesis, migration, phagocytosis and cytokinesis, and the dysregulation of Rho GTPases is associated with numerous human diseases and disorders.

scholarly journals Rho Family of Ras-Like GTPases in Early-Branching Animals

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2279
Author(s):  
Silvestar Beljan ◽  
Maja Herak Bosnar ◽  
Helena Ćetković
Keyword(s):  
Current Knowledge ◽  
Molecular Switches ◽  
Cellular Responses ◽  
Small Gtpases ◽  
Rho Family Gtpases ◽  
History Of ◽  
Rho Family ◽  
Major Branch ◽  
External Signals ◽  
Insight Into

Non-bilaterian animals consist of four phyla; Porifera, Cnidaria, Ctenophora, and Placozoa. These early-diverging animals are crucial for understanding the evolution of the entire animal lineage. The Rho family of proteins make up a major branch of the Ras superfamily of small GTPases, which function as key molecular switches that play important roles in converting and amplifying external signals into cellular responses. This review represents a compilation of the current knowledge on Rho-family GTPases in non-bilaterian animals, the available experimental data about their biochemical characteristics and functions, as well as original bioinformatics analysis, in order to gain a general insight into the evolutionary history of Rho-family GTPases in simple animals.

scholarly journals Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Michelle Seiko Lu ◽  
David G. Drubin
Keyword(s):  
Cell Migration ◽  
Genetic Analysis ◽  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Cytoskeletal Remodeling ◽  
Cell Divisions ◽  
Rho Family

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.

scholarly journals Competitive Inhibitors of Ras Effector Binding

2019 ◽  
Author(s):  
Svenja Wiechmann ◽  
Pierre Maisonneuve ◽  
Britta M. Grebbin ◽  
Meike Hoffmeister ◽  
Manuel Kaulich ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Growth Arrest ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Effector Proteins ◽  
Ras Signaling ◽  
Downstream Effector ◽  
Competitive Inhibitors ◽  
Tumorigenic Cells ◽  
Effector Binding

AbstractThe small GTPases H, K and NRas are molecular switches that are indispensable for the correct regulation of cellular proliferation and growth. Mutations in Ras are associated with cancer and result in unwanted activation of signaling processes caused by aberrant recruitment of downstream effector proteins. In this study, we have engineered variants of the Ras-binding domain (RBD) of CRAF kinase that bind with highly improved affinity the effector binding site of Ras. Structural characterization demonstrates how the engineered RBD variants outcompete effector binding and inhibit Ras signaling in cells leading to apoptosis, growth arrest and senescence. The optimized RBD variants provide new insights in Ras biology and enabled the functional stratification of Ras dependency in patient-derived colorectal cancer organoids.One sentence summaryInhibition of effector kinase binding to Ras induces senescence in non-tumorigenic cells and reveals Ras dependency in patient-derived cancer organoids.

scholarly journals Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

2019 ◽  
Author(s):  
Michelle S. Lu ◽  
David G. Drubin
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Rho Gtpase ◽  
Molecular Switches ◽  
Cell Types ◽  
Eukaryotic Cell ◽  
Small Gtpases ◽  
Cytoskeletal Remodeling ◽  
Rho Family

AbstractSmall GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none involved in the maintenance of nuclear envelope integrity or endoplasmic reticulum (ER) morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission, and by genetic analysis, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.SummaryThe small Rho GTPase Cdc42 is a well-known regulator of cytoskeletal rearrangement and polarity development in all eukaryotic cell types. Here, Lu and Drubin report the serendipitous discovery of a novel Cdc42-ESCRT-nuclear envelope/endoplasmic reticulum connection.

scholarly journals Structural basis for Rab8a GTPase recruitment of RILPL2 via LRRK2 phosphorylation of switch 2

2019 ◽  
Author(s):  
Dieter Waschbüsch ◽  
Elena Purlyte ◽  
Prosenjit Pal ◽  
Emma McGrath ◽  
Dario R. Alessi ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Effector Proteins ◽  
Structural Basis ◽  
Rab Gtpases ◽  
Post Translational Modification ◽  
Serine Threonine Kinase ◽  
Dynamic Regulation ◽  
Threonine Kinase ◽  
Membrane Protrusions ◽  
General Mode

AbstractRab8a GTPase is associated with the dynamic regulation of membrane protrusions in polarized cells. Rab8a is one of several Rab-family GTPases that are substrates of leucine-rich repeat kinase 2 (LRRK2), a serine/threonine kinase that is linked to inherited Parkinson’s disease. Rab8a is phosphorylated at T72 (pT72) in its switch 2 helix and the post-translational modification facilitates phospho-Rab8a (pRab8a) interactions with RILPL2, which subsequently regulates ciliogenesis. Here we report the crystal structure of pRab8a in complex with the phospho-Rab binding domain of RILPL2. The complex is a heterotetramer with RILPL2 forming a central α-helical dimer that bridges two pRab8a molecules. The N-termini of the α-helices cross over to form an X-shaped cap (X-cap) that enables electrostatic interactions between Arg residues from RILPL2 and the phosphate moiety from pT72. RILPL2 residues in the X-cap that are critical for pRab8a binding are conserved in the RILP family of effector proteins. We find that JIP3 and JIP4 also interact specifically with LRRK2-phosphorylated Rab10, suggesting a general mode of recognition for phosphorylated Rab GTPases by phospho-specific effectors.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

scholarly journals The Epichloë festucae Antifungal Protein Efe-AfpA Is Also a Possible Effector Protein Required for the Interaction of the Fungus with its Host Grass Festuca rubra subsp. rubra

2021 ◽  
Vol 9 (1) ◽  
pp. 140
Author(s):  
Ruying Wang ◽  
Simin Luo ◽  
Bruce B. Clarke ◽  
Faith C. Belanger
Keyword(s):  
Disease Resistance ◽  
Insect Resistance ◽  
Effector Proteins ◽  
Antifungal Protein ◽  
Grass Species ◽  
Effector Protein ◽  
Festuca Rubra ◽  
Symbiotic Interaction ◽  
Red Fescue ◽  
Epichloë Festucae

Strong creeping red fescue (Festuca rubra subsp. rubra) is a commercially important low-maintenance turfgrass and is often naturally infected with the fungal endophyte Epichloë festucae. Epichloë spp. are endophytes of several cool-season grass species, often conferring insect resistance to the grass hosts due to the production of toxic alkaloids. In addition to insect resistance, a unique feature of the strong creeping red fescue/E. festucae symbiosis is the endophyte-mediated disease resistance to the fungal pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Such disease resistance is not a general feature of other grass/ Epichloë interactions. E. festucae isolates infecting red fescue have an antifungal protein gene Efe-afpA, whereas most other Epichloë spp. do not have a similar gene. The uniqueness of this gene suggests it may, therefore, be a component of the unique disease resistance seen in endophyte-infected red fescue. Here, we report the generation of CRISPR-Cas9 Efe-afpA gene knockouts with the goal of determining if absence of the protein in endophyte-infected Festuca rubra leads to disease susceptibility. However, it was not possible to infect plants with the knockout isolates, although infection was possible with the wild type E. festucae and with complemented isolates. This raises the interesting possibility that, in addition to having antifungal activity, the protein is required for the symbiotic interaction. The antifungal protein is a small secreted protein with high expression in planta relative to its expression in culture, all characteristics consistent with effector proteins. If Efe-AfpA is an effector protein it must be specific to certain interactions, since most Epichloë spp. do not have such a gene in their genomes.

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