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Neutrophils are the primary cells recruited to inflamed sites during an innate immune response to tissue damage and/or infection. They are finely sensitive to inciting stimuli to reach in great numbers and within minutes areas of inflammation and tissue insult. For this effective response, they can detect extracellular chemical gradients and move towards higher concentrations, the so-called chemotaxis process or guided cell migration. This directed neutrophil recruitment is orchestrated by chemoattractants, a chemically diverse group of molecular guidance cues (e.g., lipids, N-formylated peptides, complement, anaphylotoxins and chemokines). Neutrophils respond to these guidance signals in a hierarchical manner and, based on this concept, they can be further subdivided into two groups: “end target” and “intermediary” chemoattractants, the signals of the former dominant over the latter. Neutrophil chemoattractants exert their effects through interaction with heptahelical G protein-coupled receptors (GPCRs) expressed on cell surfaces and the chemotactic response is mainly regulated by the Rho family of GTPases. Additionally, neutrophil behavior might differ and be affected in different complex scenarios such as disease conditions and type of vascular bed in specific organs. Finally, there are different mechanisms to disrupt neutrophil chemotaxis either associated to the resolution of inflammation or to bacterial escape and systemic infection. Therefore, in the present review, we will discuss the different molecular players involved in neutrophil chemotaxis, paying special attention to the different chemoattractants described and the way that they interact intra- and extravascularly for neutrophils to properly reach the target tissue.

Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3fT22N) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.

Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H⁺-adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acid-sensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtained in vitro and in human cells suggest that amino acid signaling begins within the lysosomal lumen. These results identify the v-ATPase as a component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing.

The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1l, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer.

Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxia-inducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.

Auxin-binding protein 1 (ABP1) was discovered nearly 40 years ago and was shown to be essential for plant development and morphogenesis, but its mode of action remains unclear. Here, we report that the plasma membrane–localized transmembrane kinase (TMK) receptor–like kinases interact with ABP1 and transduce auxin signal to activate plasma membrane–associated ROPs [Rho-like guanosine triphosphatases (GTPase) from plants], leading to changes in the cytoskeleton and the shape of leaf pavement cells in Arabidopsis. The interaction between ABP1 and TMK at the cell surface is induced by auxin and requires ABP1 sensing of auxin. These findings show that TMK proteins and ABP1 form a cell surface auxin perception complex that activates ROP signaling pathways, regulating nontranscriptional cytoplasmic responses and associated fundamental processes.

Modifications of DNA strands and nucleobases—both induced and accidental—are associated with unfavorable consequences including loss or gain in genetic information and mutations. Therefore, DNA repair proteins have essential roles in keeping genome fidelity. Recently, mounting evidence supports that 8-oxoguanine (8-oxoG), one of the most abundant genomic base modifications generated by reactive oxygen and nitrogen species, along with its cognate repair protein 8-oxoguanine DNA glycosylase1 (OGG1), has distinct roles in gene expression through transcription modulation or signal transduction. Binding to 8-oxoG located in gene regulatory regions, OGG1 acts as a transcription modulator, which can control transcription factor homing, induce allosteric transition of G-quadruplex structure, or recruit chromatin remodelers. In addition, post-repair complex formed between OGG1 and its repair product-free 8-oxoG increases the levels of active small GTPases and induces downstream signaling cascades to trigger gene expressions. The present review discusses how cells exploit damaged guanine base(s) and the authentic repair protein to orchestrate a profile of various transcriptomes in redox-regulated biological processes.

Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.

Endosomal sorting machineries regulate the transport of their cargoes among intracellular compartments. However, the molecular nature of such intracellular trafficking processes in pathogenic fungal development and pathogenicity remains unclear. Here, we dissect the roles and molecular mechanisms of two sorting nexin proteins and their cargoes in endosomal recycling in Fusarium graminearum using high-resolution microscopy and high-throughput co-immunoprecipitation strategies. We show that the sorting nexins, FgSnx41 and FgSnx4, interact with each other and assemble into a functionally interdependent heterodimer through their respective BAR domains. Further analyses demonstrate that the dimer localizes to the early endosomal membrane and coordinates endosomal sorting. The small GTPase FgRab5 regulates the correct localization of FgSnx41–FgSnx4 and is consequently required for its trafficking function. The protein FgSnc1 is a cargo of FgSnx41–FgSnx4 and regulates the fusion of secreted vesicles with the fungal growing apex and plasma membrane. In the absence of FgSnx41 or FgSnx4, FgSnc1 is missorted and degraded in the vacuole, and null deletion of either component causes defects in the fungal polarized growth and virulence. Overall, for the first time, our results reveal the mechanism of FgSnc1 endosomal recycling by FgSnx41–FgSnx4 heterodimer which is essential for polarized growth and pathogenicity in F. graminearum.