Locking GTPases covalently in their functional states

Wiegandt, David Luis; Vieweg, Sophie; Hofmann, Frank T.; Koch, Daniel; Li, Fu; Wu, Yao‐Wen; Itzen, Aymelt; Müller, Matthias; Goody, Roger S.

doi:10.1038/ncomms8773

Cited by 23 publications

(23 citation statements)

References 47 publications

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“…Similar considerations also apply to already described derivatives that have a reactive group attached to the exocyclic N2 position of the guanine base 27 . Sterically more interesting are the 5′-carbon, while still retaining the phosphorylated hydroxyl group, or the 4′ carbon.…”

Section: Discussionmentioning

confidence: 67%

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

Müller

Jeganathan

Heidrich

et al. 2017

Sci Rep

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Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRasG12C using GDP equipped with an electrophilic group at the β-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such β-modified derivatives for Ras of at least 104 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.

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Section: Discussionmentioning

confidence: 67%

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

Müller

Jeganathan

Heidrich

et al. 2017

Sci Rep

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“…5). 86 rab-10 deletion mutants displayed a decrease in the number and size of intestinal GFP:: ATG-9 foci (Fig. 5A to C), suggesting that RAB-10 is responsible for the recruitment of GFP::ATG-9 to vesicular structures, or potentially in the retrieval step from autophagosomes.…”

Section: Gtpase Cycling Of Rab-10 Is Required For Gfp::atg-9 Traffic mentioning

confidence: 99%

The recycling endosome protein RAB-10 promotes autophagic flux and localization of the transmembrane protein ATG-9

et al. 2017

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Macroautophagy/autophagy involves the formation of an autophagosome, a double-membrane vesicle that delivers sequestered cytoplasmic cargo to lysosomes for degradation and recycling. Closely related, endocytosis mediates the sorting and transport of cargo throughout the cell, and both processes are important for cellular homeostasis. However, how endocytic proteins functionally intersect with autophagy is not clear. Mutations in the DAF-2/insulin-like IGF-1 (INSR) receptor at the permissive temperature result in a small increase in GFP::LGG-1 foci, i.e. autophagosomes, but a large increase at the nonpermissive temperature, allowing us to control the level of autophagy. In a RNAi screen for endocytic genes that alter the expression of GFP::LGG-1 in daf-2 mutants, we identified RAB-10, a small GTPase that regulates basolateral endocytosis. Loss of rab-10 in daf-2 mutants results in more GFP::LGG-1-positive foci at the permissive, but less GFP::LGG-1 or SQST-1::GFP foci at the nonpermissive temperature. As previously reported, loss of rab-10 alone resulted in an increase of GFP:LGG-1 foci. Exposure of rab-10 mutant animals to chloroquine, a known inhibitor of autophagic flux, failed to increase the number of GFP::LGG-1 foci. Moreover, colocalization between LMP-1::tagRFP and GFP::LGG-1 (the lysosome and autophagosome reporters) was decreased in daf-2; rab-10 dauers at the nonpermissive temperature. Intriguingly, RAB-10 was required to maintain the normal size of GFP::ATG-9-positive structures in daf-2 mutants at both the permissive and nonpermissive temperature. Finally, we found that RAB-10 GTPase cycling was required to control the size of GFP::ATG-9 foci. Collectively, our data support a model where rab-10 controls autophagic flux by regulating autophagosome formation and maturation.

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“…This involved the use of GTP and GDP derivatives that are covalently attached to Rab proteins. Derivatives of guanine nucleotides were prepared that harbor an acrylamide residue attached via a linker to the exocyclic amino group of the guanine base (Wiegandt et al, 2015); see Figure 2. Positions were identified in Rab sequences (right,pdb ID 4PHG).…”

Section: The Mechanism Of Rab Targetingmentioning

confidence: 99%

Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport

Goody

Müller

2016

Biological Chemistry

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Our understanding of the manner in which Rab proteins regulate intracellular vesicular transport has progressed remarkably in the last one or two decades by application of a wide spectrum of biochemical, biophysical and cell biological methods, augmented by the methods of chemical biology. Important additional insights have arisen from examination of the manner in which certain bacteria can manipulate vesicular transport mechanisms. The progress in these areas is summarized here.

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Locking GTPases covalently in their functional states

Cited by 23 publications

References 47 publications

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

Nucleotide based covalent inhibitors of KRas can only be efficient in vivo if they bind reversibly with GTP-like affinity

The recycling endosome protein RAB-10 promotes autophagic flux and localization of the transmembrane protein ATG-9

Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport

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