Kyoung-in Cho

Association of the Kinesin-Binding Domain of RanBP2 to KIF5B and KIF5C Determines Mitochondria Localization and Function

The Ran‐binding protein 2 (RanBP2) is a large mosaic protein with a pleiotropic role in cell function. Although the contribution of each partner and domain of RanBP2 to its biological functions are not understood, physiological deficits of RanBP2 downregulate glucose catabolism and energy homeostasis and lead to delocalization of mitochondria components in photosensory neurons. The kinesin‐binding domain (KBD) of RanBP2 associates selectively in the central nervous system (CNS), and directly, with the ubiquitous and CNS‐specific kinesins, KIF5B and KIF5C, respectively, but not with the highly homologous KIF5A. Here, we determine the molecular and biological bases of the selective interaction between RanBP2 and KIF5B/KIF5C. This interaction is conferred by a ∼100‐residue segment, comprising a portion of the coiled‐coil and globular tail cargo‐binding domains of KIF5B/KIF5C. A single residue conserved in KIF5B and KIF5C, but not KIF5A, confers KIF5‐isotype‐specific association with RanBP2. This interaction is also mediated by a conserved leucine‐like heptad motif present in KIF5s and KBD of RanBP2. Selective inhibition of the interaction between KBD of RanBP2 and KIF5B/KIF5C in cell lines causes perinuclear clustering of mitochondria, but not of lysosomes, deficits in mitochondrial membrane potential and ultimately, cell shrinkage. Collectively, the data provide a rationale of the KIF5 subtype‐specific interaction with RanBP2 and support a novel kinesin‐dependent role of RanBP2 in mitochondria transport and function. The data also strengthen a model whereby the selection of a large array of cargoes for transport by a restricted number of motor proteins is mediated by adaptor proteins such as RanBP2.

RANBP2 is an allosteric activator of the conventional kinesin‐1 motor protein, KIF5B, in a minimal cell‐free system

The association of cargoes to kinesins is thought to promote kinesin activation, yet the validation of such a model with native cargoes is lacking because none is known to activate kinesins directly in an in vitro system of purified components. The RAN‐binding protein 2 (RANBP2), through its kinesin‐binding domain (KBD), associates in vivo with kinesin‐1, KIF5B/KIF5C. Here, we show that KBD and its flanking domains, RAN GTPase‐binding domains 2 and 3 (RBD2/RBD3), activate the ATPase activity of KIF5B approximately 30‐fold in the presence of microtubules and ATP. The activation kinetics of KIF5B by RANBP2 is biphasic and highly cooperative. Deletion of one of its RBDs lowers the activation of KIF5B threefold and abolishes cooperativity. Remarkably, RBD2–KBD–RBD3 induces unfolding and modest activation of KIF5B in the absence of microtubules. Hence, RANBP2 is the first native and positive allosteric activator known to jump‐start and boost directly the activity of a kinesin.

Haploinsufficiency of RanBP2 is neuroprotective against light-elicited and age-dependent degeneration of photoreceptor neurons

Prolonged light exposure is a determinant factor in inducing neurodegeneration of photoreceptors by apoptosis. Yet, the molecular bases of the pathways and components triggering this cell death event are elusive. Here, we reveal a prominent age-dependent increase in the susceptibility of photoreceptor neurons to undergo apoptosis under light in a mouse model. This is accompanied by light-induced subcellular changes of photoreceptors, such as dilation of the disks at the tip of the outer segments, prominent vesiculation of nascent disks, and autophagy of mitochondria into large multilamellar bodies. Notably, haploinsufficiency of Ran-binding protein-2 (RanBP2) suppresses apoptosis and most facets of membrane dysgenesis observed with age upon light-elicited stress. RanBP2 haploinsufficiency promotes decreased levels of free fatty acids in the retina independent of light exposure and turns the mice refractory to weight gain on a high-fat diet, whereas light promotes an increase in hydrogen peroxide regardless of the genotype. These studies demonstrate the presence of age-dependent and RanBP2-mediated pathways modulating membrane biogenesis of the outer segments and light-elicited neurodegeneration of photoreceptors. Furthermore, the findings support a mechanism whereby the RanBP2-dependent production of free fatty acids, metabolites thereof or the modulation of a cofactor dependent on any of these, promote apoptosis of photoreceptors in concert with the light-stimulated production of reactive oxygen species.

Ranbp2 haploinsufficiency mediates distinct cellular and biochemical phenotypes in brain and retinal dopaminergic and glia cells elicited by the Parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Many components and pathways transducing multifaceted and deleterious effects of stress stimuli remain ill-defined. The Ran-binding protein 2 (RanBP2) interactome modulates the expression of a range of clinical and cell-context-dependent manifestations upon a variety of stressors. We examined the role of Ranbp2 haploinsufficiency on cellular and metabolic manifestations linked to tyrosine-hydroxylase (TH+) dopaminergic neurons and glial cells of the brain and retina upon acute challenge to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonian neurotoxin, which models facets of Parkinson disease. MPTP led to stronger akinetic parkinsonism and slower recovery in Ranbp2+/− than wild-type mice without viability changes of brain TH+-neurons of either genotype, with the exception of transient nuclear atypia via changes in chromatin condensation of Ranbp2+/− TH+-neurons. Conversely, the number of wild-type retinal TH+-amacrine neurons compared to Ranbp2+/− underwent milder declines without apoptosis followed by stronger recoveries without neurogenesis. These phenotypes were accompanied by a stronger rise of EdU+-proliferative cells and non-proliferative gliosis of GFAP+-Müller cells in wild-type than Ranbp2+/− that outlasted the MPTP-insult. Finally, MPTP-treated wild-type and Ranbp2+/− mice present distinct metabolic footprints in the brain or selective regions thereof, such as striatum, that are supportive of RanBP2-mediated regulation of interdependent metabolic pathways of lysine, cholesterol, free-fatty acids, or their β-oxidation. These studies demonstrate contrasting gene-environment phenodeviances and roles of Ranbp2 between dopaminergic and glial cells of the brain and retina upon oxidative stress-elicited signaling and factors triggering a continuum of metabolic and cellular manifestations and proxies linked to oxidative stress, and chorioretinal and neurological disorders such as Parkinson.

Neuroprotection resulting from insufficiency of RANBP2 is associated with the modulation of protein and lipid homeostasis of functionally diverse but linked pathways in response to oxidative stress

SUMMARY Oxidative stress is a deleterious stressor associated with a plethora of disease and aging manifestations, including neurodegenerative disorders, yet very few factors and mechanisms promoting the neuroprotection of photoreceptor and other neurons against oxidative stress are known. Insufficiency of RAN-binding protein-2 (RANBP2), a large, mosaic protein with pleiotropic functions, suppresses apoptosis of photoreceptor neurons upon aging and light-elicited oxidative stress, and promotes age-dependent tumorigenesis by mechanisms that are not well understood. Here we show that, by downregulating selective partners of RANBP2, such as RAN GTPase, UBC9 and ErbB-2 (HER2; Neu), and blunting the upregulation of a set of orphan nuclear receptors and the light-dependent accumulation of ubiquitylated substrates, light-elicited oxidative stress and Ranbp2 haploinsufficiency have a selective effect on protein homeostasis in the retina. Among the nuclear orphan receptors affected by insufficiency of RANBP2, we identified an isoform of COUP-TFI (Nr2f1) as the only receptor stably co-associating in vivo with RANBP2 and distinct isoforms of UBC9. Strikingly, most changes in proteostasis caused by insufficiency of RANBP2 in the retina are not observed in the supporting tissue, the retinal pigment epithelium (RPE). Instead, insufficiency of RANBP2 in the RPE prominently suppresses the light-dependent accumulation of lipophilic deposits, and it has divergent effects on the accumulation of free cholesterol and free fatty acids despite the genotype-independent increase of light-elicited oxidative stress in this tissue. Thus, the data indicate that insufficiency of RANBP2 results in the cell-type-dependent downregulation of protein and lipid homeostasis, acting on functionally interconnected pathways in response to oxidative stress. These results provide a rationale for the neuroprotection from light damage of photosensory neurons by RANBP2 insufficiency and for the identification of novel therapeutic targets and approaches promoting neuroprotection.

Gene and protein expression pilot profiling and biomarkers in an experimental mouse model of hypertensive glaucoma.

Glaucoma is a group of genetically heterogeneous neurodegenerative disorders causing the degeneration of the ganglion neurons of the retina. Increased intraocular pressure (IOP) is a hallmark risk factor promoting the death of ganglion neurons of the retina in glaucoma. Yet, the molecular processes underlying the degeneration of these neurons by increased IOP are not understood. To gain insight into the early molecular events and discover biomarkers induced by IOP, we performed gene and protein expression profiling to compare retinas of eyes with and without high IOP in a rodent model of experimental glaucoma. This pilot study found that the IOP-mediated changes in the transcription levels of a restricted set of genes implicated in peroxisomal and mitochondrial function, modulation of neuron survival and inflammatory processes, were also accompanied by changes in the levels of proteins encoded by the same genes. With the exception of the inflammatory markers, serum amyloid-A1 (SAA1) and serum amyloid-A2 (SAA2), the IOP-induced changes in protein expression were restricted to ganglion neurons of the retina and they were detected also in the vitreous, thus suggesting an early IOP-mediated loss of ganglion cell integrity. Interestingly, SAA1 and SAA2 were induced in retinal microglia cells, whereas they were reduced in sera of IOP-responsive mice. Hence, this study defines novel IOP-induced molecular processes, biomarkers and sources thereof, and it further validates the extension of the analyses herein reported to other genes modulated by IOP.

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein–protein and protein–phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBDn = 1–4) with close structural homology to the PH domain of Brutons tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure–function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260 000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

Loss of Ranbp2 in motor neurons causes the disruption of nucleocytoplasmic and chemokine signaling and proteostasis of hnRNPH3 and Mmp28, and the development of amyotrophic lateral sclerosis (ALS)-like syndromes

ABSTRACT The pathogenic drivers of sporadic and familial motor neuron disease (MND), such amyotrophic lateral sclerosis (ALS), are unknown. MND impairs the Ran GTPase cycle, which controls nucleocytoplasmic transport, ribostasis and proteostasis; however, cause-effect mechanisms of Ran GTPase modulators in motoneuron pathobiology have remained elusive. The cytosolic and peripheral nucleoporin Ranbp2 is a crucial regulator of the Ran GTPase cycle and of the proteostasis of neurological disease-prone substrates, but the roles of Ranbp2 in motoneuron biology and disease remain unknown. This study shows that conditional ablation of Ranbp2 in mouse Thy1 motoneurons causes ALS syndromes with hypoactivity followed by hindlimb paralysis, respiratory distress and, ultimately, death. These phenotypes are accompanied by: a decline in the nerve conduction velocity, free fatty acids and phophatidylcholine of the sciatic nerve; a reduction in the g-ratios of sciatic and phrenic nerves; and hypertrophy of motoneurons. Furthermore, Ranbp2 loss disrupts the nucleocytoplasmic partitioning of the import and export nuclear receptors importin β and exportin 1, respectively, Ran GTPase and histone deacetylase 4. Whole-transcriptome, proteomic and cellular analyses uncovered that the chemokine receptor Cxcr4, its antagonizing ligands Cxcl12 and Cxcl14, and effector, latent and activated Stat3 all undergo early autocrine and proteostatic deregulation, and intracellular sequestration and aggregation as a result of Ranbp2 loss in motoneurons. These effects were accompanied by paracrine and autocrine neuroglial deregulation of hnRNPH3 proteostasis in sciatic nerve and motoneurons, respectively, and post-transcriptional downregulation of metalloproteinase 28 in the sciatic nerve. Mechanistically, our results demonstrate that Ranbp2 controls nucleocytoplasmic, chemokine and metalloproteinase 28 signaling, and proteostasis of substrates that are crucial to motoneuronal homeostasis and whose impairments by loss of Ranbp2 drive ALS-like syndromes. Summary: Loss of Ranbp2 in spinal motoneurons drives ALS syndromes in mice and Ranbp2 functions in nucleocytoplasmic trafficking, proteostasis and chemokine signaling uncover novel therapeutic targets and mechanisms for motoneuron disease.

Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms

Summary Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR1–19 and RPGRORF15 isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1&agr;1 expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1&agr;1 self-aggregation ensue. RPGR1–19 localizes to the endoplasmic reticulum, whereas RPGRORF15 presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1&agr;1. Disease mutations in RPGR1–19, RPGRORF15, or RID of RPGRIP1&agr;1, singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1&agr;1 with RPGR1–19 or RPGRORF15 in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGRORF15, but not RPGR1–19, protects the RID of RPGRIP1&agr;1 from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1&agr;1, with deficits in RPGRORF15-dependent intracellular localization of RPGRIP1&agr;1 contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies.

Uncoupling phototoxicity‐elicited neural dysmorphology and death by insidious function and selective impairment of Ran‐binding protein 2 (Ranbp2)

Morphological disintegration of neurons is coupled invariably to neural death. In particular, disruption of outer segments of photoreceptor neurons triggers photoreceptor death regardless of the pathological stressors. We show that Ranbp2−/−::Tg‐Ranbp2CLDm‐HA mice with mutations in SUMO‐binding motif (SBM) of cyclophilin‐like domain (CLD) of Ran‐binding protein 2 (Ranbp2) expressed in a null Ranbp2 background lack untoward effects in photoreceptors in the absence of light‐stress. However, compared to wild type photoreceptors, light‐stress elicits profound disintegration of outer segments of Ranbp2−/−::Tg‐Ranbp2CLDm‐HA with paradoxical age‐dependent resistance of photoreceptors to death and genotype‐independent activation of caspases. Ranbp2−/−::Tg‐Ranbp2CLDm‐HA exhibit photoreceptor death‐independent changes in ubiquitin–proteasome system (UPS), but death‐dependent increase of ubiquitin carrier protein 9(ubc9) levels. Hence, insidious functional impairment of SBM of Ranbp2s CLD promotes neuroprotection and uncoupling of photoreceptor degeneration and death against phototoxicity.