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Colorectal cancer (CRC) is the third most commonly diagnosed type of cancer worldwide and is responsible for numerous deaths. 5-fluorouracil (5-FU) is an effective chemotherapy drug commonly used in the treatment of CRC, either as monotherapy or in combination with other drugs. However, half of CRC cases are resistant to 5-FU-based therapies. To contribute to the understanding of the mechanisms underlying CRC resistance or recurrence after 5-FU-based therapies, we performed a comprehensive study integrating in silico, in vitro, and in vivo approaches. We identified differentially expressed genes and enrichment of pathways associated with recurrence after 5-FU-based therapies. Using these bioinformatics data as a starting point, we selected a group of drugs that restored 5-FU sensitivity to 5-FU resistant cells. Interestingly, treatment with the novel Rac1 inhibitor, 1A-116, reversed morphological changes associated with 5-FU resistance.. Moreover, our in vivo studies have shown that 1A-116 affected tumor growth and the development of metastasis. All our data allowed us to postulate that targeting Rac1 represents a promising avenue for the development of new treatments for patients with CRC resistant to 5-FU-based therapies. [ABSTRACT FROM AUTHOR]

Small GTPases comprise a superfamily of over 167 proteins in the human genome and are critical regulators of a variety of pathways including cell migration and proliferation. Despite the importance of these proteins in cell signaling, a standardized approach for controlling small GTPase activation within living cells is lacking. Herein, we report a split‐protein‐based approach to directly activate small GTPase signaling in living cells. Importantly, our fragmentation site can be applied across the small GTPase superfamily. We highlight the utility of these standardized parts by demonstrating the ability to directly modulate the activity of four different small GTPases with user‐defined inputs, providing the first plug and play system for direct activation of small GTPases in living cells. [ABSTRACT FROM AUTHOR]

Cilia are slender, micrometer‐long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so‐called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N‐terminal domain of CEP43 forms a homo‐dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N‐terminal domain of CEP43 from the C‐terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2‐CEP19 associates with the IFT‐B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT‐B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled‐coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia. [ABSTRACT FROM AUTHOR]

Small GTPases act as molecular switches and control numerous cellular processes by their binding and hydrolysis of guanosine triphosphate (GTP). The activity of small GTPases is coordinated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Recent structural and functional studies have characterized a subset of GAPs whose catalytic units consist of longin domains. Longin domain containing GAPs regulate small GTPases that facilitate nutrient signalling, autophagy, vesicular trafficking and lysosome homeostasis. All known examples in this GAP family function as part of larger multiprotein complexes. The three characterized mammalian protein complexes in this class are FLCN:FNIP, GATOR1 and C9orf72:SMCR8. Each complex carries out a unique cellular function by regulating distinct small GTPases. In this article, we explore the roles of longin domain GAPs in nutrient sensing, membrane dynamic, vesicular trafficking and disease. Through a structural lens, we examine the mechanism of each longin domain GAP and highlight potential therapeutic applications.

Guanine nucleotide exchange factors (GEFs) regulate the activation of small GTPases (G proteins) of the Ras superfamily proteins controlling cellular functions. Ras superfamily proteins act as 'molecular switches' that are turned 'ON' by guanine exchange. There are five major groups of Ras family GTPases: Ras, Ran, Rho, Rab and Arf, with a variety of different GEFs regulating their GTP loading. GEFs have been implicated in various diseases including cancer. This makes GEFs attractive targets to modulate signaling networks controlled by small GTPases. In this review, the roles and mechanisms of GEFs in malignancy are outlined. The mechanism of guanine exchange activity by GEFs on a small GTPase is illustrated. Then, some examples of GEFs that are significant in cancer are presented with a discussion on recent progress in therapeutic targeting efforts using a variety of approaches. Recently, GEFs have emerged as potential therapeutic targets for novel cancer drug development. Targeting small GTPases is challenging; thus, targeting their activation by GEFs is a promising strategy. Most GEF-targeted drugs are still in preclinical development. A deeper biological understanding of the underlying mechanisms of GEF activity and utilizing advanced technology are necessary to enhance drug discovery for GEFs in cancer. [ABSTRACT FROM AUTHOR]

Filamentous pathogens that cause plant diseases such as powdery mildew, rust, anthracnose, and late blight continue to represent an enormous challenge for farmers worldwide. Interestingly, these pathogens, although phylogenetically distant, initiate pathogenesis in a very similar way by penetrating the cell wall and establishing a feeding structure inside the plant host cell. To prevent pathogen ingress, the host cell responds by forming defence structures known as papillae and encasements that are thought to mediate pre- and post-invasive immunity, respectively. This form of defence is evolutionarily conserved in land plants and is highly effective and durable against a broad selection of non-adapted filamentous pathogens. As most pathogens have evolved strategies to overcome the defences of only a limited range of host plants, the papilla/encasement response could hold the potential to become an optimal transfer of resistance from one plant species to another. In this review I lay out current knowledge of the involvement of membrane trafficking that forms these important defence structures and highlight some of the questions that still need to be resolved. [ABSTRACT FROM AUTHOR]

Small GTPases are monomeric (approximately 21 kDa) proteins that regulate a number of biological processes, such as vesicle transport, cell division cycle, cell migration, invasion, adhesion, proliferation, and DNA repair. They are also involved in carcinogenesis and neurodegenerative diseases. Some of these proteins, such as the Rho family proteins, are essential regulators of actin cytoskeleton, controlling cell adhesion and motility. This review examines both normal and pathological processes in human cells that are regulated by small GTPases of the Rho family. Particular attention is paid to small GTPase inhibitors and their use in the treatment of various diseases. [ABSTRACT FROM AUTHOR]

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high‐fat diet (HFD)/low‐fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development. [ABSTRACT FROM AUTHOR]

Clathrin‐mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop‐2 as a potent CME inhibitor, we and others have reported on substantial clathrin‐independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop‐2, termed RVD‐127, to clarify Pitstop‐2 diverse effects. Using RVD‐127, we were able to trace additional protein targets of Pitstop‐2. Besides inhibiting CME, Pitstop‐2 and RVD‐127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)‐like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop‐2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop‐2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop‐2 and RVD‐127 open up novel avenues. [ABSTRACT FROM AUTHOR]

The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux. RAB21 depletion also increases lysosome function. Notably, RAB21 depletion does not overtly affect retrograde transport of IGF2R/CI-M6PR or WLS from endosomes to the trans-Golgi network. We speculate that RAB21 regulates fission of retromer-decorated endosomal tubules, as RAB21 depletion causes accumulation of the SNX27-containing retromer complex on enlarged endosomes at the perinuclear region. Functionally, RAB21 depletion sensitizes cancer cells to energy stress and inhibits tumor growth in vivo, suggesting an oncogenic role for RAB21. Overall, our study illuminates the role of RAB21 in regulating endosomal dynamics and maintaining cellular energy homeostasis and suggests RAB21 as a potential metabolic target for cancer therapy. [ABSTRACT FROM AUTHOR]

Small GTPases orchestrate numerous cellular pathways, acting as molecular switches and regulatory hubs to transmit molecular signals and because of this, they are often the target of pathogens. During infection, pathogens manipulate host cellular networks using post‐translational modifications (PTMs). AMPylation, the modification of proteins with AMP, has been identified as a common PTM utilized by pathogens to hijack GTPase signalling during infection. AMPylation is primarily carried out by enzymes with a filamentation induced by cyclic‐AMP (Fic) domain. Modification of small GTPases by AMP renders GTPases impervious to upstream regulatory inputs, resulting in unregulated downstream effector outputs for host cellular processes. Here, we overview Fic‐mediated AMPylation of small GTPases by pathogens and other related PTMs catalysed by Fic enzymes on GTPases. [ABSTRACT FROM AUTHOR]

Small GTPases play essential roles in the organization of eukaryotic cells. In recent years, it has become clear that their intracellular functions result from intricate biochemical networks of the GTPase and their regulators that dynamically bind to a membrane surface. Due to the inherent complexities of their interactions, however, revealing the underlying mechanisms of action is often difficult to achieve from in vivo studies. This review summarizes in vitro reconstitution approaches developed to obtain a better mechanistic understanding of how small GTPase activities are regulated in space and time. [ABSTRACT FROM AUTHOR]

Neurons process real-time information from axon terminals to coordinate gene expression, growth, and plasticity. Inputs from distal axons are encoded as a stream of endocytic organelles, termed signalling endosomes, targeted to the soma. Formation of these organelles depends on target-derived molecules, such as brain-derived neurotrophic factor (BDNF), which is recognised by TrkB receptors on the plasma membrane, endocytosed, and transported to the cell body along the microtubules network. Notwithstanding its physiological and neuropathological importance, the mechanism controlling the sorting of TrkB to signalling endosomes is currently unknown. In this work, we use primary mouse neurons to uncover the small GTPase Rab10 as critical for TrkB sorting and propagation of BDNF signalling from axon terminals to the soma. Our data demonstrate that Rab10 defines a novel membrane compartment that is rapidly mobilised towards the axon terminal upon BDNF stimulation, enabling the axon to fine-tune retrograde signalling depending on BDNF availability at the synapse. These results help clarifying the neuroprotective phenotype recently associated to Rab10 polymorphisms in Alzheimer’s disease and provide a new therapeutic target to halt neurodegeneration.

Steroid hormones and growth factors control neuritogenesis through their cognate receptors under physiological and pathological conditions. We have already shown that nerve growth factor and androgens induce neurite outgrowth of PC12 cells through a reciprocal crosstalk between the NGF receptor, TrkA and the androgen receptor. Here, we report that androgens or NGF induce neuritogenesis in PC12 cells through inactivation of RhoA. Ectopic expression of the dominant negative RhoA N19 promotes, indeed, the neurite-elongation of unchallenged and androgen- or NGF-challenged PC12 cells and the increase in the expression levels of βIII tubulin, a specific neuronal marker. Pharmacological inhibition of the Ser/Thr kinase ROCK, an RhoA effector, induces neuritogenesis in unchallenged PC12 cells, and potentiates the effect of androgens and NGF, confirming the role of RhoA/ROCK axis in the neuritogenesis induced by androgen and NGF, through the phosphorylation of Akt. These findings suggest that therapies based on new selective androgen receptor modulators and/or RhoA/ROCK inhibitors might exert beneficial effects in the treatment of neuro-disorders, neurological diseases and ageing-related processes. [ABSTRACT FROM AUTHOR]

Neurodegeneration is associated with defects in cytoskeletal dynamics and dysfunctions of the vesicular trafficking and sorting systems. In the last few decades, studies have demonstrated that the key regulators of cytoskeletal dynamics are proteins from the Rho family GTPases, meanwhile, the central hub for vesicle sorting and transport between target membranes is the Rab family of GTPases. In this regard, the role of Rho and Rab GTPases in the induction and maintenance of distinct functional and morphological neuronal domains (such as dendrites and axons) has been extensively studied. Several members belonging to these two families of proteins have been associated with many neurodegenerative disorders ranging from dementia to motor neuron degeneration. In this analysis, we attempt to present a brief review of the potential crosstalk between the Rab and Rho family members in neurodegenerative pathologies such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease, and amyotrophic lateral sclerosis (ALS). [ABSTRACT FROM AUTHOR]

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthesis pathway, is responsible for the production of geranylgeranyl pyrophosphate (GGPP). GGPP serves as a substrate for the post‐translational modification (geranylgeranylation) of proteins, including those belonging to the Ras superfamily of small GTPases. These proteins play key roles in signalling pathways, cytoskeletal regulation and intracellular transport, and in the absence of the prenylation modification, cannot properly localise and function. Aberrant expression of GGDPS has been implicated in various human pathologies, including liver disease, type 2 diabetes, pulmonary disease and malignancy. Thus, this enzyme is of particular interest from a therapeutic perspective. Here, we review the physiological function of GGDPS as well as its role in pathophysiological processes. We discuss the current GGDPS inhibitors under development and the therapeutic implications of targeting this enzyme. [ABSTRACT FROM AUTHOR]

Primary cilia, microtubule‐based hair‐like structures protruding from most cells, contain membranes enriched in signaling molecules and function as sensory and regulatory organelles critical for development and tissue homeostasis. Intraflagellar transport (IFT), cilia‐specific bidirectional transport, is required for the assembly, maintenance, and function of cilia. BBSome, the coat complex, acts as the adaptor between the IFT complex and membrane proteins and is therefore essential for establishing the specific compartmentalization of signaling molecules in the cilia. Recent findings have revealed that three ciliary Rab‐like small GTPases, IFT27, IFT22, and Rabl2, play critical regulatory roles in ciliary BBSome transport. In this review, we provide an overview of these three Rab‐like small GTPases and their relationship with BBSome. [ABSTRACT FROM AUTHOR]

RAC1 is a highly conserved Rho GTPase critical for many cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Some of these variants have previously been shown to have a dominant negative effect. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. Here, we describe eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblast spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo , these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborization neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61–R68 within the switch II region of RAC1 cause a developmental syndrome. Our findings reveal that these variants cause altered downstream signalling, resulting in abnormal neuronal morphology and reveal the WAVE regulatory complex/Arp2/3 pathway as a possible therapeutic target for activating RAC1 variants. These insights also have the potential to inform the mechanism and therapy for other disorders caused by variants in genes encoding other Rho GTPases, their regulators and downstream effectors. [ABSTRACT FROM AUTHOR]

Intercellular communication is an essential process in all multicellular organisms. During this process, molecules secreted by one cell will bind to a receptor on the cognate cell leading to the subsequent uptake of the receptor‐ligand complex. Once inside, the cell then determines the fate of the receptor‐ligand complex and any other proteins that were endocytosed together. Approximately 80% of endocytosed material is recycled back to the plasma membrane either directly or indirectly via the Golgi apparatus and the remaining 20% is delivered to the lysosome for degradation. Although most pathways have been identified, we still lack understanding on how specificity in sorting of recycling cargos into different pathways is achieved, and how the cell reaches high accuracy of these processes in the absence of clear sorting signals in the bulk of the client proteins. In this review, we will summarize our current understanding of the mechanism behind recycling cargo sorting and propose a model of differential affinities between cargo and cargo receptors/adaptors with regards to iterative sorting in endosomes. [ABSTRACT FROM AUTHOR]

Complex N-glycans are asparagine (N)-linked branched sugar chains attached to secretory proteins in eukaryotes. They are produced by modification of N-linked oligosaccharide structures in the endoplasmic reticulum (ER) and Golgi apparatus. Complex N-glycans formed in the Golgi apparatus are often assigned specific roles unique to the host organism, with their roles in plants remaining largely unknown. Using inhibitor (kifunensine, KIF)-hypersensitivity as read-out, we identified Arabidopsis mutants that require complex N-glycan modification. Among over 100 KIF-sensitive mutants, one showing abnormal secretory organelles and a salt-sensitive phenotype contained a point mutation leading to amino-acid replacement (G69R) in ARFA1E, a small Arf1-GTPase family protein presumably involved in vesicular transport. In-vitro assays showed that the G69R exchange interferes with protein activation. In vivo, ARFA1EG69R caused dominant-negative effects, altering the morphology of the ER, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transports (endocytosis/endocytic recycling) of essential glycoprotein KORRIGAN1, one of KIF-sensitivity targets, is slowed down constitutively as well as under salt stress in ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, ARFA1EG69R mutant established a link between KIF-targeted luminal glycoprotein functions/dynamics and cytosolic regulators of vesicle transport in endosome-/cell wall-associated tolerance mechanisms., (© The Author(s) 2025. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

MicroRNAs (miRNAs) are emerging as effective therapeutic agents. When testing whether miR-145-5p could alleviate kidney injury, we unexpectedly found that extracellular vesicles loaded with miR-145-5p induced proteinuria and podocyte foot process effacement in normal control mice. To explore the mechanism of miR-145-5p’s toxicity to podocytes, we hypothesized that miR-145-5p could enter podocytes and inhibit genes essential for podocytes. We demonstrated that systemically administered miRNA can enter podocytes. Next, we predicted 611 podocyte essential genes based on single-cell RNA sequencing (RNA-seq) and found that 32 of them are predicted to be targeted by miR-145-5p. Functional annotation of the 32 podocyte essential genes revealed small GTPase-mediated signal transduction as the top pathway. We experimentally validated that miR-145-5p targeted Arhgap24 and Srgap1, the essential regulators of the Rho family of small GTPases, increased the activity of Rac1 and Cdc42, and reduced RhoA activity, accompanied by cellular injury, in podocytes. These results explain how miR-145-5p has deleterious effect on podocytes. Most importantly, our study provides a novel approach to investigate how a miRNA affects a given cell type, allowing not only identification of the molecular mechanism underlying an observed side effect of a miRNA drug but also prediction of miRNA drug toxicity on various cell types.

Neurodegeneration is associated with defects in cytoskeletal dynamics and dysfunctions of the vesicular trafficking and sorting systems. In the last few decades, studies have demonstrated that the key regulators of cytoskeletal dynamics are proteins from the Rho family GTPases, meanwhile, the central hub for vesicle sorting and transport between target membranes is the Rab family of GTPases. In this regard, the role of Rho and Rab GTPases in the induction and maintenance of distinct functional and morphological neuronal domains (such as dendrites and axons) has been extensively studied. Several members belonging to these two families of proteins have been associated with many neurodegenerative disorders ranging from dementia to motor neuron degeneration. In this analysis, we attempt to present a brief review of the potential crosstalk between the Rab and Rho family members in neurodegenerative pathologies such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington disease, and amyotrophic lateral sclerosis (ALS).

Rac-GEFs operate in a nonredundant manner as downstream effectors of receptor tyrosine kinases to promote ruffle formation, indicative of unique modes of regulation and targeting. Current research is shedding light on the intricate signaling paradigms shaping spatiotemporal activation of the small GTPase Rac during the generation of actin-rich membrane protrusions. [ABSTRACT FROM AUTHOR]

RasGRPs are guanine nucleotide exchange factors that are specific for Ras or Rap, and are important regulators of cellular signaling. Aberrant expression or mutation of RasGRPs results in disease. An analysis of RasGRP1 SNP variants led to the conclusion that the charge of His 212 in RasGRP1 alters signaling activity and plasma membrane recruitment, indicating that His 212 is a pH sensor that alters the balance between the inactive and active forms of RasGRP1. To understand the structural basis for this effect we compared the structure of autoinhibited RasGRP1, determined previously, to those of active RasGRP4:H-Ras and RasGRP2:Rap1b complexes. The transition from the autoinhibited to the active form of RasGRP1 involves the rearrangement of an inter-domain linker that displaces inhibitory inter-domain interactions. His 212 is located at the fulcrum of these conformational changes, and structural features in its vicinity are consistent with its function as a pH-dependent switch.

Abstract The pathogenic mechanisms underlying the development of Alzheimer’s disease (AD) remain elusive and to date there are no effective prevention or treatment for AD. Farnesyltransferase (FT) catalyzes a key posttranslational modification process called farnesylation, in which the isoprenoid farnesyl pyrophosphate is attached to target proteins, facilitating their membrane localization and their interactions with downstream effectors. Farnesylated proteins, including the Ras superfamily of small GTPases, are involved in regulating diverse physiological and pathological processes. Emerging evidence suggests that isoprenoids and farnesylated proteins may play an important role in the pathogenesis of AD. However, the dynamics of FT and protein farnesylation in human brains and the specific role of neuronal FT in the pathogenic progression of AD are not known. Here, using postmortem brain tissue from individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimer’s dementia, we found that the levels of FT and membrane-associated H-Ras, an exclusively farnesylated protein, and its downstream effector ERK were markedly increased in AD and MCI compared with NCI. To elucidate the specific role of neuronal FT in AD pathogenesis, we generated the transgenic AD model APP/PS1 mice with forebrain neuron-specific FT knockout, followed by a battery of behavioral assessments, biochemical assays, and unbiased transcriptomic analysis. Our results showed that the neuronal FT deletion mitigates memory impairment and amyloid neuropathology in APP/PS1 mice through suppressing amyloid generation and reversing the pathogenic hyperactivation of mTORC1 signaling. These findings suggest that aberrant upregulation of protein farnesylation is an early driving force in the pathogenic cascade of AD and that targeting FT or its downstream signaling pathways presents a viable therapeutic strategy against AD.

Abstract Background Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. Results Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. Conclusions Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Cardiac hypertrophy results from the heart reacting and adapting to various pathological stimuli and its persistent development is a major contributing factor to heart failure. However, the molecular mechanisms of cardiac hypertrophy remain unclear. Small GTPases in the Ras, Rho, Rab, Arf and Ran subfamilies exhibit GTPase activity and play crucial roles in regulating various cellular responses. Previous studies have shown that Ras, Rho and Rab are closely linked to cardiac hypertrophy and that their overexpression can induce cardiac hypertrophy. Here, we review the functions of small GTPases in cardiac hypertrophy and provide additional insights and references for the prevention and treatment of cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Members of the Ras superfamily have been found to perform several functions leading to the development of eukaryotes. These small GTPases are divided into five major subfamilies, and their regulators can "turn on" and "turn off" signals. Recent studies have shown that this superfamily of proteins has various roles in the process of vascular development, such as vasculogenesis and angiogenesis. Here, we discuss the role of these subfamilies in the development of the vascular system in zebrafish. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming represents a hallmark of tumorigenesis to sustain survival in harsh conditions, rapid growth and metastasis in order to resist to cancer therapies. These metabolic alterations involve glucose metabolism, known as the Warburg effect, increased glutaminolysis and enhanced amino acid and lipid metabolism, especially the cholesterol biosynthesis pathway known as the mevalonate pathway and these are upregulated in several cancer types, including acute myeloid leukemia (AML). In particular, it was demonstrated that the mevalonate pathway has a pivotal role in cellular transformation. Therefore, targeting this biochemical process with drugs such as statins represents a promising therapeutic strategy to be combined with other anticancer treatments. In the last decade, several studies have revealed that amino-bisphosphonates (BP), primarily used for bone fragility disorders, also exhibit potential anti-cancer activity in leukemic cells, as well as in patients with symptomatic multiple myeloma. Indeed, these compounds inhibit the farnesyl pyrophosphate synthase, a key enzyme in the mevalonate pathway, reducing isoprenoid formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. This, in turn, inhibits the prenylation of small Guanosine Triphosphate-binding proteins, such as Ras, Rho, Rac, Rab, which are essential for regulating cell survival membrane ruffling and trafficking, interfering with cancer key signaling events involved in clonal expansion and maturation block of progenitor cells in myeloid hematological malignancies. Thus, in this review, we discuss the recent advancements about bisphosphonates' effects, especially zoledronate, analyzing the biochemical mechanisms and anti-tumor effects on AML model systems. Future studies will be oriented to investigate the clinical relevance and significance of BP treatment in AML, representing an attractive therapeutic strategy that could be integrated into chemotherapy. [ABSTRACT FROM AUTHOR]

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP‐loading and abundance of Ras and Rheb in lipidated protein‐enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non‐canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract Ran (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other functions in the cytoplasm, as well as in cellular transformation when switched on. Unlike other members of the GTPase superfamily, Ran binds more tightly to GDP than to GTP due to the presence of an auto-inhibitory C-terminal tail. Multiple missense mutations in the C-terminus of Ran occur in cancers, but their biological significance remains unclear. Here, the quantitative GDP/GTP binding preference of four engineered mutations with unstable C-termini was analyzed using a devised mant-GDP dissociation assay. The results showed that the impact of different C-terminal mutations depends on multiple factors. Although these mutants were more GTP-loaded in human cells, they were shown to be more cytoplasmic, and to support nuclear transport with minimally or partially reduced efficiency. Further, several Ran cancer mutants were compromised in autoinhibition, slightly more GTP-bound, more cytoplasmic, and enhanced the proliferation of A549 and HeLa cells in vitro. Thus, our work reveals a new route of Ran activation independent of guanine nucleotide exchange factor (GEF), which may account for the hyper-proliferation induced by Ran cancer mutations.

The RAS family of small GTPases is mutated in roughly a fifth of human cancers. Hotspot point mutations at codons G12, G13, and Q61 account for 95% of all these mutations, which are well established to render the encoded proteins oncogenic. In humans, this family comprises three genes: HRAS, NRAS, and KRAS. Accumulating evidence argues that oncogenic RAS point mutations may be initiating, as they are often truncal in human tumours and capable of inducing tumorigenesis in mice. As such, there is great interest in detecting oncogenic mutation in the RAS genes to understand the origins of cancer, as well as for early detection purposes. To this end, we previously adapted the microbial ultra-sensitive Maximum Depth Sequencing (MDS) assay for the murine Kras gene, which was capable of detecting oncogenic mutations in the tissues of mice days after carcinogen exposure, essentially capturing the very first step in tumour initiation. Given this, we report here the adaption and details of this assay to detect mutations in a human KRAS sequence at an analytic sensitivity of one mutation in a million independently barcoded templates. This humanized version of MDS can thus be exploited to detect oncogenic mutations in KRAS at an incredible sensitivity and modified for the same purpose for the other RAS genes. [ABSTRACT FROM AUTHOR]

Summary: Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.

The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs). [ABSTRACT FROM AUTHOR]

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD. • Enhanced temperature causes ectopic activation of Rho GTPases in MKD fibroblasts. • Cell cycle-related genes are downregulated in MKD fibroblasts. • Actin cytoskeleton-related genes are upregulated in MKD fibroblasts. • MKD affects cell cycle progression and actin cytoskeleton dynamics. • The expression of glycolytic genes is increased in MKD fibroblasts. [ABSTRACT FROM AUTHOR]

Among the numerous oncogenes involved in human cancers, KRAS represents the most studied and best characterized cancer-related genes. Several therapeutic strategies targeting oncogenic KRAS (KRASonc) signaling pathways have been suggested, including the inhibition of synthetic lethal interactions, direct inhibition of KRASonc itself, blockade of downstream KRASonc effectors, prevention of post-translational KRASonc modifications, inhibition of the induced stem cell-like program, targeting of metabolic peculiarities, stimulation of the immune system, inhibition of inflammation, blockade of upstream signaling pathways, targeted RNA replacement, and oncogene-induced senescence. Despite intensive and continuous efforts, KRASonc remains an elusive target for cancer therapy. To highlight the progress to date, this review covers a collection of studies on therapeutic strategies for KRAS published from 1995 to date. An overview of the path of progress from earlier to more recent insights highlight novel opportunities for clinical development towards KRASonc-signaling targeted therapeutics.

Small GTPases act as molecular switches in regulating a myriad of cellular signaling, cytoskeletal dynamics, vesicular trafficking, and membrane/organelle transport processes. Here, I provide an editorial overview of papers collected in this Special Issue on the “Regulation and Function of Small GTPases 2.0”.

Plexins are semaphorin receptors that play essential roles in mammalian neuronal axon guidance and in many other important mammalian biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism and is modulated by small GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations, we showed that RND1 reinforces the plexin dimerization interface, whereas RhoD destabilizes it due to their differential interaction with the cell membrane. Upon binding plexin at the Rho-GTPase-binding domain (RBD), RND1 and RhoD interact differently with the inner leaflet of the cell membrane and exert opposite effects on the dimerization interface via an allosteric network involving the RBD, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively charged membrane interface.