Search Results (33)

Although LRRK2 mutations modulate systemic glucose homeostasis and metabolic dysfunction precedes Parkinson’s disease (PD) motor symptoms; the way in which pathogenic variants of LRRK2 disrupt astrocytic glucose metabolism and organellar homeostasis remains poorly understood. Here, we demonstrate that LRRK2-I1371V mutation causes profound metabolic and organellar dysfunction in LRRK2-I1371V PD-iPSC-derived astrocytes and U87 cells overexpressing I1371V variant. LRRK2-I1371V astrocytes exhibit significantly reduced GLUT1 expression and cell surface localization, resulting in impaired glucose uptake and decreased lactate production. This metabolic insufficiency correlates with cascading mitochondrial dysfunction, characterized by membrane depolarization, elevated reactive oxygen species, enhanced ubiquitination and reduced proteasomal activity. Reduced LAMP1/LAMP2 expression, impaired lysosomal acidification, and selective cathepsin D deficiency were observed. Accumulation of undegraded cargo was confirmed by transmission electron microscopy upon α-synuclein exposure. ER stress was evident by upregulation of GADD34/CHOP, increased phospho-PERK, and reduced nascent protein synthesis. Increased ER–mitochondrial contact via MAMs and enhanced STIM1-ORAI3 clustering reflect compensatory but ultimately insufficient responses to energy stress. Our results reveal that LRRK2-I1371V induces glucose uptake deficits, leading to energy depletion and integrated ER–mitochondria–lysosome dysfunction, thus indicating restoration of astrocytic metabolic capacity as a potential therapeutic strategy for LRRK2-associated PD. Full article

Mutations in leucine-rich repeat kinase 2 (LRRK2) are among the most common genetic causes of Parkinson’s disease (PD), yet substantial heterogeneity exists among pathogenic variants. How mutations in distinct functional domains of LRRK2 differentially perturb cellular homeostasis remains incompletely understood. Here, we compared two pathogenic LRRK2 mutations—G2019S in the kinase domain and I1371V in the GTPase domain—across multiple cellular models, including SH-SY5Y and U87 cells, and healthy human iPSC-derived floor plate cells. We demonstrate that the I1371V mutation induces markedly more severe cellular dysfunction than G2019S. I1371V-expressing cells exhibited elevated LRRK2 autophosphorylation at S1292 and robust hyperphosphorylation of Rab8A and Rab10, indicating enhanced downstream signaling. These alterations impaired sterol trafficking, leading to selective depletion of membrane cholesterol without changes in total cellular cholesterol. Consequently, I1371V cells displayed increased membrane fluidity, disrupted microdomain organization, altered membrane topology, reduced caveolin-1 expression, and impaired dopamine transporter surface expression and dopamine uptake. Lipidomic profiling further revealed a broad disruption of lipid homeostasis, including reductions in cholesteryl esters, sterols, sphingolipids, and glycerophospholipids, whereas G2019S cells showed comparatively modest changes. Pharmacological intervention revealed mutation-specific responses, with the non-selective LRRK2 modulator GW5074 outperforming the kinase-selective inhibitor MLi-2 in restoring Rab8A phosphorylation, membrane integrity, and dopaminergic function. Collectively, these findings identify membrane lipid dysregulation as a central cell biological mechanism in LRRK2-associated PD and underscore the importance of variant-specific therapeutic strategies. Full article

The essential SUP35 gene encodes yeast translation termination factor Sup35/eRF3. The N-terminal domain of Sup35 is also responsible for Sup35 prionization that leads to generation of the [PSI⁺] prion. Previously we isolated different types of sup35 mutations (missense and nonsense) and demonstrated that sup35 nonsense mutations (sup35-n) are incompatible with the [PSI⁺] prion, leading to lethality of sup35-n [PSI⁺] haploid cells. Here, we show that sup35 missense mutations (sup35-m) within conservative regions of the Sup35 C-domain result in lethality of [PSI⁺] cells because of weak activity of Sup35/eRF3 as a translation termination factor. Mutant Sup35 maintain their ability to be incorporated into pre-existing [PSI⁺] aggregates and to form amyloid aggregates in vitro, while sup35-m mutations do not influence the [PSI⁺] prion induction and stability. All these mutations (D363N, R372K, T378I) are located in the conservative GTPase region of Sup35, decreasing the GTPase activity of mutated proteins. We propose that such low activity of mutant Sup35 combined with aggregation of Sup35 constituting the [PSI⁺] prion is not sufficient to maintain the viability of yeast cells. Full article

Clostridioides difficile Infection (CDI) continues to be a major cause of antibiotic-associated diarrhea and pseudomembranous colitis, fueled in large measure by virulence factors TcdA and TcdB. These giant glucosyltransferase toxins interfere with host cytoskeletal integrity and inflammatory signaling by inhibiting Rho GTPase; however, the detailed structural dynamics, receptor selectivity, and subcellular trafficking mechanisms remain in part unspecified. This review integrates recent insights from cryo-electron microscopy (cryo-EM) and X-ray crystallography to describe the quaternary architecture of TcdA/B, emphasizing conformational changes key to pore formation and endosomal escape. We also examine the genomic heterogeneity of hypervirulent C. difficile strains (e.g., ribotype 027), correlating toxin gene polymorphisms (e.g., tcdC mutations) with increased toxin production and virulence. Mechanistic explanations of toxin-driven inflammasome activation and epithelial barrier dysfunction are situated within host immune evasion mechanisms, including microbiota-derived bile acid regulation of toxin stability. Subsequent innovative therapeutic strategies, encompassing the utilization of engineered neutralizing antibodies that specifically target the autoprocessing domain alongside structure-guided small-molecule inhibitors, are subjected to a rigorous evaluation. By integrating structural biology, systems-level omics, and clinical epidemiology, this review establishes a comprehensive framework for understanding C. difficile toxin pathogenesis and guiding next-generation precision antimicrobials. Full article

The transmembrane protein β-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer’s disease (AD). The β-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic β-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD. Full article

Transient homo-dimerization of the RAS GTPase at the plasma membrane has been shown to promote the mitogen-activated protein kinase (MAPK) signaling pathway essential for cell proliferation and oncogenesis. To date, numerous crystallographic studies have focused on the well-defined GTPase domains of RAS isoforms, which lack the disordered C-terminal membrane anchor, thus providing limited structural insight into membrane-bound RAS molecules. Recently, lipid-bilayer nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses have revealed several distinct structures of the membrane-anchored homodimers of KRAS, an isoform that is most frequently mutated in human cancers. The KRAS dimerization interface is highly plastic and altered by biologically relevant conditions, including oncogenic mutations, the nucleotide states of the protein, and the lipid composition. Notably, PRE-derived structures of KRAS homodimers on the membrane substantially differ in terms of the relative orientation of the protomers at an “α–α” dimer interface comprising two α4–α5 regions. This interface plasticity along with the altered orientations of KRAS on the membrane impact the accessibility of KRAS to downstream effectors and regulatory proteins. Further, nanodisc platforms used to drive KRAS dimerization can be used to screen potential anticancer drugs that target membrane-bound RAS dimers and probe their structural mechanism of action. Full article

GATOR1 (GAP Activity TOward Rag 1) is an evolutionarily conserved GTPase-activating protein complex that controls the activity of mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid availability in cells. Genetic mutations in the GATOR1 subunits, NPRL2 (nitrogen permease regulator-like 2), NPRL3 (nitrogen permease regulator-like 3), and DEPDC5 (DEP domain containing 5), have been associated with epilepsy in humans; however, the specific effects of these mutations on GATOR1 function and mTORC1 regulation are not well understood. Herein, we report that epilepsy-linked mutations in the NPRL2 subunit of GATOR1, NPRL2-L105P, -T110S, and -D214H, increase basal mTORC1 signal transduction in cells. Notably, we show that NPRL2-L105P is a loss-of-function mutation that disrupts protein interactions with NPRL3 and DEPDC5, impairing GATOR1 complex assembly and resulting in high mTORC1 activity even under conditions of amino acid deprivation. Furthermore, our studies reveal that the GATOR1 complex is necessary for the rapid and robust inhibition of mTORC1 in response to growth factor withdrawal or pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). In the absence of the GATOR1 complex, cells are refractory to PI3K-dependent inhibition of mTORC1, permitting sustained translation and restricting the nuclear localization of TFEB, a transcription factor regulated by mTORC1. Collectively, our results show that epilepsy-linked mutations in NPRL2 can block GATOR1 complex assembly and restrict the appropriate regulation of mTORC1 by canonical PI3K-dependent growth factor signaling in the presence or absence of amino acids. Full article

Intracellular endosomal trafficking controls the balance between protein degradation and synthesis, i.e., proteostasis, but also many of the cellular signaling pathways that emanate from activated growth factor receptors after endocytosis. Endosomal trafficking, sorting, and motility are coordinated by the activity of small GTPases, including Rab proteins, whose function as molecular switches direct activity at endosomal membranes through effector proteins. Rab7 is particularly important in the coordination of the degradative functions of the pathway. Rab7 effectors control endosomal maturation and the properties of late endosomal and lysosomal compartments, such as coordination of recycling, motility, and fusion with downstream compartments. The spatiotemporal regulation of endosomal receptor trafficking is particularly challenging in neurons because of their enormous size, their distinct intracellular domains with unique requirements (dendrites vs. axons), and their long lifespans as postmitotic, differentiated cells. In Charcot–Marie–Tooth 2B disease (CMT2B), familial missense mutations in Rab7 cause alterations in GTPase cycling and trafficking, leading to an ulcero-mutilating peripheral neuropathy. The prevailing hypothesis to account for CMT2B pathologies is that CMT2B-associated Rab7 alleles alter endocytic trafficking of the neurotrophin NGF and its receptor TrkA and, thereby, disrupt normal trophic signaling in the peripheral nervous system, but other Rab7-dependent pathways are also impacted. Here, using TrkA as a prototypical endocytic cargo, we review physiologic Rab7 effector interactions and control in neurons. Since neurons are among the largest cells in the body, we place particular emphasis on the temporal and spatial regulation of endosomal sorting and trafficking in neuronal processes. We further discuss the current findings in CMT2B mutant Rab7 models, the impact of mutations on effector interactions or balance, and how this dysregulation may confer disease. Full article

Owing to the presence of multiple enzymatic domains, LRRK2 has been associated with a diverse set of cellular functions and signaling pathways. It also has several pathological mutant-variants, and their incidences show ethnicity biases and drug-response differences with expression in dopaminergic-neurons and astrocytes. Here, we aimed to assess the cell-intrinsic effect of the LRRK2-I1371V mutant variant, prevalent in East Asian populations, on astrocyte yield and biology, involving Nrf2-mediated glutathione machinery, glutamate uptake and metabolism, and ATP generation in astrocytes derived from LRRK2-I1371V PD patient iPSCs and independently confirmed in LRRK2-I1371V-overexpressed U87 cells. Astrocyte yield (GFAP-immunopositive) was comparable between LRRK2-I1371V and healthy control (HC) populations; however, the astrocytic capability to mitigate oxidative stress in terms of glutathione content was significantly reduced in the mutant astrocytes, along with a reduction in the gene expression of the enzymes involved in glutathione machinery and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Simultaneously, a significant decrease in glutamate uptake was observed in LRRK2-I1371V astrocytes, with lower gene expression of glutamate transporters SLC1A2 and SLC1A3. The reduction in the protein expression of SLC1A2 was also directly confirmed. Enzymes catalyzing the generation of γ glutamyl cysteine (precursor of glutathione) from glutamate and the metabolism of glutamate to enter the Krebs cycle (α-ketoglutaric acid) were impaired, with significantly lower ATP generation in LRRK2-I1371V astrocytes. De novo glutamine synthesis via the conversion of glutamate to glutamine was also affected, indicating glutamate metabolism disorder. Our data demonstrate for the first time that the mutation in the LRRK2-I1371V allele causes significant astrocytic dysfunction with respect to Nrf2-mediated antioxidant machinery, AT -generation, and glutamate metabolism, even with comparable astrocyte yields. Full article

Mutations in the multidomain protein Leucine-rich-repeat kinase 2 (LRRK2) have been identified as a genetic risk factor for both sporadic and familial Parkinson’s disease (PD). LRRK2 has two enzymatic domains: a RocCOR tandem with GTPase activity and a kinase domain. In addition, LRRK2 has three N-terminal domains: ARM (Armadillo repeat), ANK (Ankyrin repeat), and LRR (Leucine-rich-repeat), and a C-terminal WD40 domain, all of which are involved in mediating protein–protein interactions (PPIs) and regulation of the LRRK2 catalytic core. The PD-related mutations have been found in nearly all LRRK2 domains, and most of them have increased kinase activity and/or decreased GTPase activity. The complex activation mechanism of LRRK2 includes at least intramolecular regulation, dimerization, and membrane recruitment. In this review, we highlight the recent developments in the structural characterization of LRRK2 and discuss these developments from the perspective of the LRRK2 activation mechanism, the pathological role of the PD mutants, and therapeutic targeting. Full article

Mitochondrial dysfunction or morphological abnormalities in human pathogenic fungi are known to contribute to azole resistance; however, the underlying molecular mechanisms are unknown. In this study, we investigated the link between mitochondrial morphology and azole resistance in Candida glabrata, which is the second most common cause of human candidiasis worldwide. The ER-mitochondrial encounter structure (ERMES) complex is thought to play an important role in the mitochondrial dynamics necessary for mitochondria to maintain their function. Of the five components of the ERMES complex, deletion of GEM1 increased azole resistance. Gem1 is a GTPase that regulates the ERMES complex activity. Point mutations in GEM1 GTPase domains were sufficient to confer azole resistance. The cells lacking GEM1 displayed abnormalities in mitochondrial morphology, increased mtROS levels, and increased expression of azole drug efflux pumps encoded by CDR1 and CDR2. Interestingly, treatment with N-acetylcysteine (NAC), an antioxidant, reduced ROS production and the expression of CDR1 in Δgem1 cells. Altogether, the absence of Gem1 activity caused an increase in mitochondrial ROS concentration, leading to Pdr1-dependent upregulation of the drug efflux pump Cdr1, resulting in azole resistance. Full article

Rab22a is an important small GTPase protein the molecule that is involved in intracellular transportation and regulation of proteins. It also plays an important role in antigens uptake, transportation, regulation of endosome morphology, and also regulates the transport of antigens to MHC (Major Histocompatibility Complex) molecules. To investigate the role of Rab22a, the intracellular co-localization of chicken Rab22a (cRab22a) molecule and its relationship to BF and chicken invariant chain (cIi) molecules was studied. A 3D protein structure of Rab22a was constructed by using informatics tools (DNASTAR 4.0 and DNAMAN). Based on the model, the corresponding recombinant eukaryotic plasmids were constructed by point mutations in the protein’s structural domains. HEK 293T cells were co-transfected with plasmids pEGFP-C1-cIi to observe the intracellular co-localization. Secondly, the DC2.4 Mouse Dendritic Cell and Murine RAW 264.7 cells were transfected with recombinant plasmids of pmCherry-cRab22a and pmCherry-mRab22a respectively. Subsequently, the intracellular localization of cRab22a in early and late endosomes was observed with specific antibodies against EEA1 and LAMP1 respectively. For gene expression-based studies, the cRab22a gene was down-regulated and up-regulated in HD11 cells, following the detection of transcription levels of the BFa (MHCIa) and cIi genes by real-time quantitative PCR (RT-qPCR). The interactions of the cRab22a gene with BFa and cIi were detected by co-immunoprecipitation (Co-IP) and Western blot. The results showed that the protein structures of chicken and mouse Rab22a were highly homologous (95.4%), and both localize to the early and late endosomes. Ser41 and Tyr74 are key amino acids in the Switch regions of Rab22a which maintain its intracellular localization. The down-regulation of cRab22a gene expression significantly reduced (p < 0.01) the transcription of BFa (MHCIa) and cIi in HD11 cells. However, when the expression of the cRab22a gene was increased 55 times as compared to control cells, the expression of the BFa (MHCIa) gene was increased 1.7 times compared to the control cells (p < 0.01), while the expression of the cIi gene did not significantly differ from control (p > 0.05). Western blot results showed that cRab22a could not directly bind to BFa and cIi. So, cRab22a can regulate BFa and cIi protein molecules indirectly. It is concluded that cRab22a was localized with cIi in the endosome. The Switch regions of cRab22a are the key domains that affect intracellular localization and colocalization of the cIi molecule. Full article

Leucine rich-repeat kinase 2 (LRRK2) is the most well-known etiologic gene for familial Parkinson’s disease (PD). Its gene product is a large kinase with multiple functional domains that phosphorylates a subset of Rab small GTPases. However, studies of autopsy cases with LRRK2 mutations indicate a varied pathology, and the molecular functions of LRRK2 and its relationship to PD pathogenesis are largely unknown. Recently, non-autonomous neurodegeneration associated with glial cell dysfunction has attracted attention as a possible mechanism of dopaminergic neurodegeneration. Molecular studies of LRRK2 in astrocytes and microglia have also suggested that LRRK2 is involved in the regulation of lysosomal and other organelle dynamics and inflammation. In this review, we describe the proposed functions of LRRK2 in glial cells and discuss its involvement in the pathomechanisms of PD. Full article

Phenotypic variations in Charcot-Marie-Tooth disease type 2A (CMT2A) result from the many mutations in the mitochondrial fusion protein, mitofusin 2 (MFN2). While the GTPase domain mutations of MFN2 lack the ability to hydrolyze GTP and complete mitochondrial fusion, the mechanism of dysfunction in HR1 domain mutations has yet to be explored. Using Mfn1/Mfn2 double null cells and Mfn2 knock out (KO) fibroblasts, we measured the ability of this variant protein to change conformations and hydrolyze GTP. We found that a mutation in the HR1 domain (M376A) of MFN2 results in conformational change dysfunction while maintaining GTPase ability. Prolonged exposure to mitofusin agonist MiM 111 reverses mitochondrial fusion dysfunction in the HR1 mutant through encouraging an open conformation, resulting in a potential therapeutic model in this variant. Herein, we describe a novel mechanism of dysfunction in MFN2 variants through exploring domain-specific mitochondrial characteristics leading to CMT2A. Full article

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42^Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42. Full article