RanBP2 Modulates Cox11 and Hexokinase I Activities and Haploinsufficiency of RanBP2 Causes Deficits in Glucose Metabolism

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2(−/−) are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2(+/−) mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies

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. RPGRIP1α1 expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α1 self-aggregation ensue. RPGR1–19 localizes to the endoplasmic reticulum, whereas RPGRORF15 presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α1. Disease mutations in RPGR1–19, RPGRORF15, or RID of RPGRIP1α1, singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α1 with RPGR1–19 or RPGRORF15 in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGRORF15, but not RPGR1–19, protects the RID of RPGRIP1α1 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 RPGRIP1α1, with deficits in RPGRORF15-dependent intracellular localization of RPGRIP1α1 contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies

Assessment of a clinical checklist in the diagnosis of fragile X syndrome in India

Fragile X syndrome (FRAXA) is one of the most common forms of mental retardation. It is caused by the expansion of cytosine-guanine-guanine (CGG) repeats in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene, located at Xq27.3. The number of CGG repeats in the FMR1 gene occurs in four distinct ranges: 2-50 (normal), 50-60 (gray zone), 60-200 (premutation), and > 200 (full mutation). When the number of CGG repeats exceeds 200, the gene becomes hypermethylated and transcriptionally silenced, which results in the loss of FMR protein and causes FRAXA. The key clinical features of FRAXA are mental retardation, macro-orchidism, long face, prominent jaw, connective tissue abnormalities, and behavioral problems. A modified 15-item checklist was used to assess the clinical features in 337 individuals (316 males and 21 females) who have mental retardation of unknown etiology. These patients were in institutions. Molecular diagnosis was performed using polymerase chain reaction and Southern blot analysis and revealed that 14 males were positive for FRAXA. Studies of the families of the affected males revealed an additional 11 affected males and 20 carrier females. Retrospective analysis of clinical features was performed in a total of 327 males and 41 females. Six clinical features were statistically significant in FRAXA individuals when compared to non-FRAXA individuals. These features were hyperactivity (p < 0.05), poor eye contact (p < 0.001), hyper extensibility of joints (p < 0.001), large ears (p < 0.001), macro-orchidism (p < 0.001), and a family history of mental retardation (p < 0.001). When a total score of 5 out of 15 was used as the threshold clinical score, 73.18% of the patients with total scores < 5 could be eliminated as FRAXA-negative patients, thereby improving the reliability of FRAXA testing using the clinical checklist

Insertion Mutagenesis of the Murine <i>RanBP2</i> Gene

<div><p>(A) Diagram of the genomic region of <i>RanBP2</i> disrupted by insertion trap mutagenesis with a bicistronic reporter vector between exon 1 and 2. The bicistronic transcript produces two proteins under regulation of RanBP2. Upon splicing of <i>RanBP2,</i> a fusion between exon 1 and β-geo (a fusion between the <i>β-gal</i> and <i>neo</i> genes) is generated, while human placental alkaline phophatase (PLAP) is independently translated using the internal ribosome entry site. Consistent with previous studies, the expression of the former is directed to cell bodies, while expression of the latter is targeted to the axonal processes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020177#pgen-0020177-b067" target="_blank">67</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020177#pgen-0020177-b068" target="_blank">68</a>]. Transcriptional 5′ RACE analysis detects a fusion between exon 1 and β-geo.</p><p>(B) Southern analysis of the <i>RanBP2</i> locus of wild-type and heterozygous genomic DNA of tails of F1 mice digested with <i>Ppu</i>MI (left panel) and <i>Hind</i>III (right panel) with probes at the 3′ (left panel) and 5′ (right panel) flanking regions of the insertion breakpoint. Q1 is a cosmid containing the <i>RanBP2</i> gene up to exon 20 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020177#pgen-0020177-b004" target="_blank">4</a>].</p><p>(C) Lateroventral view of a whole-mount stain of a ~12.5 dpc heterozygous embryo for PLAP and β-gal (inset picture) activities. Although PLAP was broadly expressed (e.g., somites, limbs, and CNS), the PLAP and β-Gal (inset picture) expression was particularly high in the optic vesicle (arrow). X-gal single (D) and combined staining with PLAP (E) of a retinal section of a 3-mo-old RanBP2^+/− mouse. Consistent with previous immunocytochemistry studies, β-Gal activity is detected in the neuroretinal bodies and inner segment compartment of photoreceptors with conspicuously strong expression in ganglion cells. PLAP expression is found throughout the plexiform/synaptic layers and outer segment of photoreceptors (E). GC, ganglion cell; PLAP, human placental alkaline phophatase; ROS, rod outer segment; RIS, rod inner segment; ONL, outer nuclear layer; OPL, outer plexiform (synaptic) layer; INL, inner nuclear layer; IPL, inner plexiform (synaptic) layer; GC, ganglion cell layer.</p></div

Metabolic Phenotypes of <i>RanBP2^+/−</i> Inbred Mice on High-Fat Diet

<div><p>(A) 3-mo-old inbred <i>RanBP2^+/−</i> mice (<i>n</i> = 5) have normal glucose clearance rates upon glucose challenge and overnight fasting.</p><p>(B) In contrast, 6-mo-old inbred <i>RanBP2^+/−</i> mice (<i>n</i> = 5) have significantly decreased glucose clearance rates upon glucose challenge and overnight fasting.</p><p>(C) Fasted 6- to 8-mo-old <i>RanBP2^+/+</i> and <i>RanBP2^+/−</i> mice have no difference in insulin-mediated glucose uptake as assayed by insulin tolerance test (<i>n</i> = 5).</p><p>(D) Pyruvate tolerance test shows normal rise in glucose but decreased glucose clearance between inbred <i>RanBP2^+/+</i> and <i>RanBP2^+/−</i> mice (<i>n</i> = 5).</p></div