Selective axonal translation of the mRNA isoform encoding prenylated Cdc42 supports axon growth

ABSTRACT The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3′ untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

Lipidation of a bacterial effector is critical for bacterial evasion of host-defense

The Rab32 antimicrobial pathway has been shown to restrict Salmonella Typhi, in mouse macrophages. The broad-host pathogen Salmonella Typhimurium however has evolved a strategy to evade the Rab32 antimicrobial pathway, via its effector protein GtgE. GtgE is a cysteine protease that specifically mediates the cleavage and inactivation of Rab32. Here we show that GtgE association and targeting to membranes is critical for its efficient proteolytic activity. The C-terminus of GtgE contains a CaaX motif, which can be post-translationally modified by the host’s prenylation machinery. Using a combination of confocal microscopy and subcellular fractionation we show that a cysteine in the CaaX motif is crucial for GtgE membrane targeting and, more importantly, GtgE localization to the Salmonella-containing vacuole. We also demonstrated that prenylation of CaaX is important for an effective and fast Rab32 cleavage, which in turn helps Salmonella to successfully survive in macrophages and establish an in vivo infection in mice. Our findings shed light on the importance of a host mediated post-translational modification that targets GtgE to the membranes where it can efficiently cleave and inactivate Rab32, leading to a better Salmonella survival in macrophages.Author summarySalmonella species includes a large group of bacteria that cause disease in different hosts. While some serovars are host generalists, others are restricted to humans. This is the case of Salmonella Typhi, responsible for Typhoid fever, a disease that affects millions globally. We have previously discovered an antimicrobial activity in macrophages that is controlled by Rab32. While the broad-host bacterium Salmonella Typhimurium effectively counteracts this mechanism through the delivery of two effectors, GtgE and SopD2, Salmonella Typhi does not express those effectors and cannot survive in mouse macrophages. In this article, we demonstrate how Salmonella Typhimurium exploits a host machinery to modify GtgE. We show that this host mediated modification is important for GtgE intracellular localization and effective Rab32 targeting, resulting in both a better intracellular survival and infection in vivo.

Cysteine residues in the C-terminal tail of connexin32 regulate its trafficking

ABSTRACTGap junctions (GJ)s are formed by the assembly of constituent transmembrane proteins called connexins (Cxs). Aberrations in this assembly of Cxs are observed in several genetic diseases as well as in cancers. Hence it becomes imperative to understand the molecular mechanisms underlying such assembly defect. The polarized cells in the epithelia express Connexin32 (Cx32). The carboxyl-terminal tail (CT) of Cx32 orchestrates several aspects of GJ dynamics, function and growth. The study here was aimed at determining if post-translational modifications, specifically, palmitoylation of cysteine residues, present in the CT of Cx32, has any effect on GJ assembly. The CT of Cx32 was found to harbor three cysteine residues, which are likely to be modified by palmitoylation. The study here has revealed for the first time that Cx32 is palmitoylated at cysteine 217 (C217). However, it was found that mutating C217 to alanine affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, it was discovered that mutating cysteine 280 and 283 in combination, blocked the transport of Cx32 from the Golgi to the cell surface. Overall, the findings reveal the importance of the two terminal cysteine residues of Cx32 in regulating its trafficking and stability and hence is ability to assemble into GJs, possibly as being part of a CAAX motif in its CT.

The Cellular Localization of the p42 and p46 Oligoadenylate Synthetase 1 Isoforms and Their Impact on Mitochondrial Respiration

The importance of the IFN-induced oligoadenylate synthetase (OAS) proteins and the OAS/RNase L pathway in the innate response against viral pathogens is well-established, however the observed differences in anti-viral activity between the human OAS1 p46 and p42 isoforms are not fully understood. The protein expression of these isoforms is determined by the SNP rs10774671, either being an A or a G allele resulting in expression of either the p42 or the p46 isoform. Using fluorescence microscopy and immunoblot analysis of fractionated cell samples, we show here that the CaaX motif is of key importance to the cellular localization. The OAS1 p42 isoform is mainly located in the cytosol, whereas the p46 isoform with a C-terminal CaaX motif is translocated to membranous organelles, like the mitochondria. We furthermore observed differences between p42 and p46 in their effect on mitochondrial physiology using high resolution respirometry and fluorometry. Overexpression of OAS1 p42 and IFN-β treatment of HeLa cells (AA genotype) resulted in significantly increased respiration, which was not seen with p46 overexpression. The difference in subcellular localization and mitochondrial effect of these two OAS1 isoforms might help to explain the anti-viral mechanisms that differentiate these proteins.

Biochemical and structural characterization of murine GBP7, a guanylate binding protein with an elongated C-terminal tail

Guanylate-binding proteins (GBPs) constitute a family of interferon-inducible guanosine triphosphatases (GTPases) that are key players in host defense against intracellular pathogens ranging from protozoa to bacteria and viruses. So far, human GBP1 and GBP5 as well as murine GBP2 (mGBP2) have been biochemically characterized in detail. Here, with murine GBP7 (mGBP7), a GBP family member with an unconventional and elongated C-terminus is analyzed. The present study demonstrates that mGBP7 exhibits a concentration-dependent GTPase activity and an apparent GTP turnover number of 20 min−1. In addition, fluorescence spectroscopy analyses reveal that mGBP7 binds GTP with high affinity (KD = 0.22 µM) and GTPase activity assays indicate that mGBP7 hydrolyzes GTP to GDP and GMP. The mGBP7 GTPase activity is inhibited by incubation with γ-phosphate analogs and a K51A mutation interfering with GTP binding. SEC-MALS analyses give evidence that mGBP7 forms transient dimers and that this oligomerization pattern is not influenced by the presence of nucleotides. Moreover, a structural model for mGBP7 is provided by homology modeling, which shows that the GTPase possesses an elongated C-terminal (CT) tail compared with the CaaX motif-containing mGBP2 and human GBP1. Molecular dynamics simulations indicate that this tail has transmembrane characteristics and, interestingly, confocal microscopy analyses reveal that the CT tail is required for recruitment of mGBP7 to the parasitophorous vacuole of Toxoplasma gondii.

Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

Stable localization of the Rheb GTPase to lysosomes is thought to be required for activation of mTOR complex 1 (mTORC1) signaling. However, the lysosome targeting mechanisms for Rheb remain unclear. We therefore investigated the relationship between Rheb subcellular localization and mTORC1 activation. Surprisingly, we found that Rheb was undetectable at lysosomes. Nonetheless, functional assays in knockout human cells revealed that farnesylation of the C-terminal CaaX motif on Rheb was essential for Rheb-dependent mTORC1 activation. Although farnesylated Rheb exhibited partial endoplasmic reticulum (ER) localization, constitutively targeting Rheb to ER membranes did not support mTORC1 activation. Further systematic analysis of Rheb lipidation revealed that weak, nonselective, membrane interactions support Rheb-dependent mTORC1 activation without the need for a specific lysosome targeting motif. Collectively, these results argue against stable interactions of Rheb with lysosomes and instead that transient membrane interactions optimally allow Rheb to activate mTORC1 signaling.

Weak Membrane Interactions Allow Rheb to Activate mTORC1 Signaling Without Major Lysosome Enrichment

Stable localization of the Rheb GTPase to lysosomes is thought to be required for activation of mTORC1 signaling. However, the lysosome targeting mechanisms for Rheb remain unclear. We therefore investigated the relationship between Rheb subcellular localization and mTORC1 activation. Surprisingly, we found that Rheb was undetectable at lysosomes. Nonetheless, functional assays in knockout human cells revealed that farnesylation of the C-terminal CaaX motif on Rheb was essential for Rheb-dependent mTORC1 activation. Although farnesylated Rheb exhibits partial endoplasmic reticulum localization, constitutively targeting Rheb to ER membranes did not support mTORC1 activation. Further systematic analysis of Rheb lipidation revealed that weak, non-selective, membrane interactions support Rheb-dependent mTORC1 activation without the need for a specific lysosome targeting motif. Collectively, these results argue against stable interactions of Rheb with lysosomes and instead that transient membrane interactions optimally allow Rheb to activate mTORC1 signaling.

GEF mechanism revealed by the structure of SmgGDS-558 and farnesylated RhoA complex and its implication for a chaperone mechanism

SmgGDS has dual functions in cells and regulates small GTPases as both a guanine nucleotide exchange factor (GEF) for the Rho family and a molecular chaperone for small GTPases possessing a C-terminal polybasic region followed by four C-terminal residues called the CaaX motif, which is posttranslationally prenylated at its cysteine residue. Our recent structural work revealed that SmgGDS folds into tandem copies of armadillo-repeat motifs (ARMs) that are not present in other GEFs. However, the precise mechanism of GEF activity and recognition mechanism for the prenylated CaaX motif remain unknown because SmgGDS does not have a typical GEF catalytic domain and lacks a pocket to accommodate a prenyl group. Here, we aimed to determine the crystal structure of the SmgGDS/farnesylated RhoA complex. We found that SmgGDS induces a significant conformational change in the switch I and II regions that opens up the nucleotide-binding site, with the prenyl group fitting into the cryptic pocket in the N-terminal ARMs. Taken together, our findings could advance the understanding of the role of SmgGDS and enable drug design strategies for targeting SmgGDS and small GTPases.

A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent phenotypes

The modifications occurring to CaaX proteins have largely been established using few reporter molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, and COOH-terminal methylation. Here, we investigated the coupling of these modifications in the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is resistant to post-isoprenylation events. These results expand on the complexity of protein isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of Ydj1p and perhaps other CaaX proteins.