Haiqing Yi

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.

RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis

The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1(tm1Tili) mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1(nmf247) that is phenotypically distinct from Rpgrip1(tm1Tili) mice. Photoreceptor degeneration in homozygous Rpgrip1(nmf247) mice is earlier in onset and more severe when compared with Rpgrip1(tm1Tili) mice. Also, ultrastructural studies reveal that whereas Rpgrip1(nmf247) mutants have a normal structure and number of connecting cilia, unlike Rpgrip1(tm1Tili) mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1(nmf247) mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.

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.

Characterization of a canine model of glycogen storage disease type IIIa.

SUMMARY Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disease caused by deficiency of glycogen debranching enzyme (GDE) in liver and muscle. The disorder is clinically heterogeneous and progressive, and there is no effective treatment. Previously, a naturally occurring dog model for this condition was identified in curly-coated retrievers (CCR). The affected dogs carry a frame-shift mutation in the GDE gene and have no detectable GDE activity in liver and muscle. We characterized in detail the disease expression and progression in eight dogs from age 2 to 16 months. Monthly blood biochemistry revealed elevated and gradually increasing serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities; serum creatine phosphokinase (CPK) activity exceeded normal range after 12 months. Analysis of tissue biopsy specimens at 4, 12 and 16 months revealed abnormally high glycogen contents in liver and muscle of all dogs. Fasting liver glycogen content increased from 4 months to 12 months, but dropped at 16 months possibly caused by extended fibrosis; muscle glycogen content continually increased with age. Light microscopy revealed significant glycogen accumulation in hepatocytes at all ages. Liver histology showed progressive, age-related fibrosis. In muscle, scattered cytoplasmic glycogen deposits were present in most cells at 4 months, but large, lake-like accumulation developed by 12 and 16 months. Disruption of the contractile apparatus and fraying of myofibrils was observed in muscle at 12 and 16 months by electron microscopy. In conclusion, the CCR dogs are an accurate model of GSD IIIa that will improve our understanding of the disease progression and allow opportunities to investigate treatment interventions.

The Cyclophilin-like Domain of Ran-binding Protein-2 Modulates Selectively the Activity of the Ubiquitin-Proteasome System and Protein Biogenesis

The ubiquitin-proteasome system (UPS) plays a critical role in protein degradation. The 19S regulatory particle (RP) of the 26S proteasome mediates the recognition, deubiquitylation, unfolding, and channeling of ubiquitylated substrates to the 20S proteasome. Several subunits of the 19S RP interact with a growing number of factors. The cyclophilin-like domain (CLD) of Ran-binding protein-2 (RanBP2/Nup358) associates specifically with at least one subunit, S1, of the base subcomplex of the 19S RP, but the functional implications of this interaction on the UPS activity are elusive. This study shows the CLD of RanBP2 promotes selectively the accumulation of a subset of reporter substrates of the UPS, such as the ubiquitin (Ub)-fusion yellow fluorescent protein (YFP) degradation substrate, UbG76V-YFP, and the N-end rule substrate, Ub-R-YFP. Conversely, the degradation of endoplasmic reticulum and misfolded proteins, and of those linked to UPS-independent degradation, is not affected by CLD. The selective effect of CLD on the UPS in vivo is independent of, and synergistic with, proteasome inhibitors, and CLD does not affect the intrinsic proteolytic activity of the 20S proteasome. The inhibitory activity of CLD on the UPS resides in a purported SUMO binding motif. We also found two RanBP2 substrates, RanGTPase-activating protein and retinitis pigmentosa GTPase regulator interacting protein-1α1, whose steady-state levels are selectively modulated by CLD. Hence, the CLD of RanBP2 acts as a novel auxiliary modulator of the UPS activity; it may contribute to the molecular and subcellular compartmentation of the turnover of properly folded proteins and modulation of the expressivity of several neurological diseases.

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.

Genomic abnormalities of the murine model of Fabry disease after disease-related perturbation, a systems biology approach

Fabry disease is a disorder of α-d-galactosyl-containing glycolipids resulting from a deficiency of α-galactosidase A. Patients have a poorly understood vascular dysregulation. We hypothesized that disease-related perturbation by using enzyme replacement therapy in the murine model of Fabry disease would provide insight into abnormal biological processes in Fabry disease. Gene expression analyses of the heart, aorta, and liver of male α-galactosidase A knockout mice 28 weeks of age were compared with that of WT mice. Microarray analyses were performed before and after six weekly injections of α-galactosidase A. Alteration of Rpgrip1 ranked highest statistically in all three organs when knockout mice were compared with WT, and its splice variants responded in a unique way to α-galactosidase A. Enzyme replacement therapy tended to not only normalize gene expression, e.g., reduce the overexpression of securin, but also specifically modified gene expression in each tissue examined. Following multiple comparison analysis, gene expression correlation graphs were constructed, and a priori hypotheses were examined by using structural equation modeling. This systems biology approach demonstrated multiple and complex parallel cellular abnormalities in Fabry disease. These abnormalities form the basis for informed, in a Bayesian sense, sequential, hypothesis-driven research that can be subsequently tested experimentally.