Proteomic Analysis of Extracellular Vesicles Derived from MDA-MB-231 Cells in Microgravity.

Patients with triple-negative breast cancer (TNBC) have a relatively poor prognosis and cannot benefit from endocrine and/or targeted therapy. Considerable effort has been devoted toward the elucidation of the molecular mechanisms and potential diagnostic/therapeutic targets. However, it is inefficient and often ineffective to study the biological nuances of TNBC in large-scale clinical trials. In contrast, the investigation of the association between molecular alterations induced through controlled variables and relevant physiochemical characteristics of TNBC cells in laboratory settings is simple, definite, and efficient in exploring the molecular mechanisms. In this study, microgravity was selected as the sole variable of study as it can inhibit cancer cell viability, proliferation, metastasis, and chemoresistance. Identifying the key molecules that shift cancer cells toward a less aggressive phenotype may facilitate future TNBC studies. We focused on extracellular vesicles (EV) derived from TNBC MDA-MB-231 cells in microgravity, which mediate intercellular communication by transporting signaling molecules between cells. Our results show that in comparison with cells in full gravity, EV release rate decreased in microgravity while average EV size increased. In addition, we found EVs may be superior to cells in analyzing differentially expressed proteins, especially those that are down-regulated ones and usually unidentified or neglected in analysis of intact cellular contents. Proteomic analysis of both EVs and cells further revealed a significant correlation with GTPases and proliferation of MDA-MB-231 cells in microgravity. Altogether, our findings would further inspire in-depth correlative cancer biological studies and subsequent clinical research.

ARF family GTPases with links to cilia.

The ADP-ribosylation factor (ARF) superfamily of regulatory GTPases, including both the ARF and ARF-like (ARL) proteins, control a multitude of cellular functions, including aspects of vesicular traffic, lipid metabolism, mitochondrial architecture, the assembly and dynamics of the microtubule and actin cytoskeletons, and other pathways in cell biology. Considering their general utility, it is perhaps not surprising that increasingly ARF/ARLs have been found in connection to primary cilia. Here, we critically evaluate the current knowledge of the roles four ARF/ARLs (ARF4, ARL3, ARL6, ARL13B) play in cilia and highlight key missing information that would help move our understanding forward. Importantly, these GTPases are themselves regulated by guanine nucleotide exchange factors (GEFs) that activate them and by GTPase-activating proteins (GAPs) that act as both effectors and terminators of signaling. We believe that the identification of the GEFs and GAPs and better models of the actions of these GTPases and their regulators will provide a much deeper understanding and appreciation of the mechanisms that underly ciliary functions and the causes of a number of human ciliopathies.

A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

ATPase and GTPase Tangos Drive Intracellular Protein Transport.

The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways.

Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells.

The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.

Crystal structure of Leishmania major ADP-ribosylation factor-like 1 and a classification of related GTPase family members in this Kinetoplastid.

ADP-ribosylation factor-like (ARL) proteins are small GTPases that undergo conformational changes upon nucleotide binding, and which regulate the affinity of ARLs for binding other proteins, lipids or membranes. There is a paucity of structural data on this family of proteins in the Kinetoplastida, despite studies implicating them in key events related to vesicular transport and regulation of microtubule-dependent processes. The crystal structure of Leishmania major ARL1 in complex with GDP has been determined to 2.1 Å resolution and reveals a high degree of structural conservation with human ADP-ribosylation factor 1 (ARF1). Putative L. major and Trypanosoma brucei ARF/ARL family members have been classified based on structural considerations, amino acid sequence conservation combined with functional data on Kinetoplastid and human orthologues. This classification may guide future studies designed to elucidate the function of specific family members.

Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans.

Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well-established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24 degrees C but are essential at 37 degrees C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post-mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post-mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence.

Protective role of interferon-induced Mx GTPases against influenza viruses.

Mx proteins are interferon-induced large GTPases with antiviral activities. They inhibit a wide range of viruses by blocking early stages of the replication cycles. Importantly, Mx GTPases also suppress the growth of highly pathogenic influenza A viruses, such as currently circulating H5N1 viruses or the pandemic H1N1 virus strain of 1918. In this paper, the authors review the properties of Mx proteins and discuss their role in host defence against highly pathogenic viruses. The authors further suggest that mammalian Mx proteins may normally provide a barrier against zoonotic transmission of avian influenza A viruses and that acquired resistance to the antiviral action of human MxA may be one factor, among many others, that facilitates the spread of pandemic strains in human populations. The presently available evidence suggests that Mx proteins of domestic chickens lack the ability to efficiently combat avian influenza viruses known to cause devastating infections in this species. The deliberate introduction of an antivirally active Mx gene originating from resistant birds or mammals may confer some degree of protection and thus stop commercial birds from serving as amplifying hosts of potentially pandemic influenza virus strains.

A systematic classification of Plasmodium falciparum P-loop NTPases: structural and functional correlation.

BACKGROUND: The P-loop NTPases constitute one of the largest groups of globular protein domains that play highly diverse functional roles in most of the organisms. Even with the availability of nearly 300 different Hidden Markov Models representing the P-loop NTPase superfamily, not many P-loop NTPases are known in Plasmodium falciparum. A number of characteristic attributes of the genome have resulted into the lack of knowledge about this functionally diverse, but important class of proteins. METHOD: In the study, protein sequences with characteristic motifs of NTPase domain (Walker A and Walker B) are computationally extracted from the P. falciparum database. A detailed secondary structure analysis, functional classification, phylogenetic and orthology studies of the NTPase domain of repertoire of 97 P. falciparum P-loop NTPases is carried out. RESULTS: Based upon distinct sequence features and secondary structure profile of the P-loop domain of obtained sequences, a cladistic classification is also conceded: nucleotide kinases and GTPases, ABC and SMC family, SF1/2 helicases, AAA+ and AAA protein families. Attempts are made to identify any ortholog(s) for each of these proteins in other Plasmodium sp. as well as its vertebrate host, Homo sapiens. A number of P. falciparum P-loop NTPases that have no homologue in the host, as well as those annotated as hypothetical proteins and lack any characteristic functional domain are identified. CONCLUSION: The study suggests a strong correlation between sequence and secondary structure profile of P-loop domains and functional roles of these proteins and thus provides an opportunity to speculate the role of many hypothetical proteins. The study provides a methodical framework for the characterization of biologically diverse NTPases in the P. falciparum genome.The efforts made in the analysis are first of its kind; and the results augment to explore the functional role of many of these proteins from the parasite that could provide leads to identify novel drug targets against malaria.

Atlastin GTPases are required for Golgi apparatus and ER morphogenesis.

The hereditary spastic paraplegias (SPG1-33) comprise a cluster of inherited neurological disorders characterized principally by lower extremity spasticity and weakness due to a length-dependent, retrograde axonopathy of corticospinal motor neurons. Mutations in the gene encoding the large oligomeric GTPase atlastin-1 are responsible for SPG3A, a common autosomal dominant hereditary spastic paraplegia. Here we describe a family of human GTPases, atlastin-2 and -3 that are closely related to atlastin-1. Interestingly, while atlastin-1 is predominantly localized to vesicular tubular complexes and cis-Golgi cisternae, mostly in brain, atlastin-2 and -3 are localized to the endoplasmic reticulum (ER) and are most enriched in other tissues. Knockdown of atlastin-2 and -3 levels in HeLa cells using siRNA (small interfering RNA) causes disruption of Golgi morphology, and these Golgi structures remain sensitive to brefeldin A treatment. Interestingly, expression of SPG3A mutant or dominant-negative atlastin proteins lacking GTPase activity causes prominent inhibition of ER reticularization, suggesting a role for atlastin GTPases in the formation of three-way junctions in the ER. However, secretory pathway trafficking as assessed using vesicular stomatitis virus G protein fused to green fluorescent protein (VSVG-GFP) as a reporter was essentially normal in both knockdown and dominant-negative overexpression conditions for all atlastins. Thus, the atlastin family of GTPases functions prominently in both ER and Golgi morphogenesis, but they do not appear to be required generally for anterograde ER-to-Golgi trafficking. Abnormal morphogenesis of the ER and Golgi resulting from mutations in atlastin-1 may ultimately underlie SPG3A by interfering with proper membrane distribution or polarity of the long corticospinal motor neurons.

The Mx GTPase family of interferon-induced antiviral proteins.

Mx proteins are interferon-induced members of the dynamin superfamily of large GTPases. They inhibit a wide range of viruses by blocking an early stage of the replication cycle. Studies in genetically defined mouse strains highlight their powerful action in early antiviral host defence.

Analysis of septins across kingdoms reveals orthology and new motifs.

BACKGROUND: Septins are cytoskeletal GTPase proteins first discovered in the fungus Saccharomyces cerevisiae where they organize the septum and link nuclear division with cell division. More recently septins have been found in animals where they are important in processes ranging from actin and microtubule organization to embryonic patterning and where defects in septins have been implicated in human disease. Previous studies suggested that many animal septins fell into independent evolutionary groups, confounding cross-kingdom comparison. RESULTS: In the current work, we identified 162 septins from fungi, microsporidia and animals and analyzed their phylogenetic relationships. There was support for five groups of septins with orthology between kingdoms. Group 1 (which includes S. cerevisiae Cdc10p and human Sept9) and Group 2 (which includes S. cerevisiae Cdc3p and human Sept7) contain sequences from fungi and animals. Group 3 (which includes S. cerevisiae Cdc11p) and Group 4 (which includes S. cerevisiae Cdc12p) contain sequences from fungi and microsporidia. Group 5 (which includes Aspergillus nidulans AspE) contains sequences from filamentous fungi. We suggest a modified nomenclature based on these phylogenetic relationships. Comparative sequence alignments revealed septin derivatives of already known G1, G3 and G4 GTPase motifs, four new motifs from two to twelve amino acids long and six conserved single amino acid positions. One of these new motifs is septin-specific and several are group specific. CONCLUSION: Our studies provide an evolutionary history for this important family of proteins and a framework and consistent nomenclature for comparison of septin orthologs across kingdoms.

Arabidopsis SHORT INTEGUMENTS 2 is a mitochondrial DAR GTPase.

The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short integuments due to early cessation of cell division in these structures. SIN2 was isolated and encodes a putative GTPase sharing features found in the novel DAR GTPase family. DAR proteins share a signature DAR motif and a unique arrangement of the four conserved GTPase G motifs. We found that DAR GTPases are present in all examined prokaryotes and eukaryotes and that they have diversified into four paralogous lineages in higher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases have been found to localize to mitochondria and are related to eubacterial proteins that facilitate essential steps in biogenesis of the large ribosomal subunit. We propose a similar role for SIN2 in mitochondria. A sin2 insertional allele has ovule effects similar to sin2-1, but more pronounced pleiotropic effects on vegetative and floral development. The diverse developmental effects of the mitochondrial SIN2 GTPase support a mitochondrial role in the regulation of multiple developmental pathways.

The homologous putative GTPases Grn1p from fission yeast and the human GNL3L are required for growth and play a role in processing of nucleolar pre-rRNA.

Grn1p from fission yeast and GNL3L from human cells, two putative GTPases from the novel HSR1_MMR1 GTP-binding protein subfamily with circularly permuted G-motifs play a critical role in maintaining normal cell growth. Deletion of Grn1 resulted in a severe growth defect, a marked reduction in mature rRNA species with a concomitant accumulation of the 35S pre-rRNA transcript, and failure to export the ribosomal protein Rpl25a from the nucleolus. Deleting any of the Grn1p G-domain motifs resulted in a null phenotype and nuclear/nucleolar localization consistent with the lack of nucleolar export of preribosomes accompanied by a distortion of nucleolar structure. Heterologous expression of GNL3L in a Deltagrn1 mutant restored processing of 35S pre-rRNA, nuclear export of Rpl25a and cell growth to wild-type levels. Genetic complementation in yeast and siRNA knockdown in HeLa cells confirmed the homologous proteins Grn1p and GNL3L are required for growth. Failure of two similar HSR1_MMR1 putative nucleolar GTPases, Nucleostemin (NS), or the dose-dependent response of breast tumor autoantigen NGP-1, to rescue deltagrn1 implied the highly specific roles of Grn1p or GNL3L in nucleolar events. Our analysis uncovers an important role for Grn1p/GNL3L within this unique group of nucleolar GTPases.

Novel small GTPase subfamily capable of associating with tubulin is required for chromosome segregation.

The small GTPase superfamily, which includes the Ras, Rho/Rac, Rab, Arf and Ran subfamilies, serves as a signal transducer to regulate cell proliferation and differentiation, actin cytoskeleton, membrane trafficking, and nuclear transport. Here, we identify novel GTPases (human Gie1 and Gie2) that form a distinct subfamily of the small GTPases in terms of their sequences and intracellular function. Gie stands for 'novel GTPase indispensable for equal segregation of chromosomes', and this subfamily is conserved in multicellular organisms. Expression of dominant-negative Gie mutants in mammalian cells or knockdown of Gie transcripts using RNA interference in Drosophila S2 cells induced abnormal morphology in the chromosome segregation. Gie protein has ability to bind to tubulin and localizes with microtubules on the spindle mid-zone in late mitosis. Furthermore, overexpression of Gie mutants that lack putative effector domains but have tubulin-binding ability induced micronucleus formation. Thus, this is the first report showing that a small GTPase subfamily capable of associating with microtubules might be involved in chromosome segregation.

Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors.

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution.

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype.

Small GTPases and the evolution of the eukaryotic cell.

The origin of eukaryotes is one of the major challenges of evolutionary cell biology. Other than the endosymbiotic origin of mitochondria and chloroplasts, the steps leading to eukaryotic endomembranes and endoskeleton are poorly understood. Ras-family small GTPases are key regulators of cytoskeleton dynamics, vesicular trafficking and nuclear function. They are specific for eukaryotes and their expansion probably traces the evolution of core eukaryote features. The phylogeny of small GTPases suggests that the first endomembranes to evolve during eukaryote evolution had secretory, and not phagocytic, function. Based on the reconstruction of putative roles for ancestral small GTPases, a hypothetical scenario on the origins of the first endomembranes, the nucleus, and phagocytosis is presented.

Mammalian septins nomenclature.

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.