Selective Impairment of a Subset of Ran-GTP-binding Domains of Ran-binding Protein 2 (Ranbp2) Suffices to Recapitulate the Degeneration of the Retinal Pigment Epithelium (RPE) Triggered by Ranbp2 Ablation

Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2^−/−, with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2^−/− background. Among the transgenic lines produced, only Tg^RBD2/3*-HA::RPE-cre::Ranbp2^−/−-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2^−/−. By contrast, Tg^RBD2/3*-HA expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2^−/− and Tg^RBD2/3*-HA::RPE-cre::Ranbp2^−/− share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.     

Functional analysis of the two cyclophilin isoforms of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

The nitrogen fixing Sinorhizobium meliloti possesses two genes, ppiA and ppiB, encoding two cyclophilin isoforms which belong to the superfamily of peptidyl prolyl cis/trans isomerases (PPIase, EC: 5.2.1.8). Here, we functionally characterize the two proteins and we demonstrate that both recombinant cyclophilins are able to isomerise the Suc-AAPF-pNA synthetic peptide but neither of them displays chaperone function in the citrate synthase thermal aggregation assay. Furthermore, we observe that the expression of both enzymes increases the viability of E. coli BL21 in the presence of abiotic stress conditions such as increased heat and salt concentration. Our results support and strengthen previous high-throughput studies implicating S. meliloti cyclophilins in various stress conditions.     

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3b^P132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3b^P132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a “helper” that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.     

The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1^−/− mice have reduced viability and fail to thrive early after birth. Male Ube2j1^−/− mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1^−/− elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.     

Gene expression and biochemical characterization of Azotobacter vinelandii cyclophilins and Protein Interaction Studies of the cytoplasmic isoform with dnaK and lpxH

The soil nitrogen-fixing bacterium Azotobacter vinelandii possesses two cyclophilins, comprising putative cytoplasmic and periplasmic isoforms, designated as AvPPIB and AvPPIA, respectively. Both recombinant cyclophilins have been purified and their peptidyl-prolyl cis/trans isomerase activity against Suc-Ala-Xaa-Pro-Phe-pNA synthetic peptides has been characterized. The substrate specificity of both cyclophilins is typical for bacterial cyclophilins, with Suc-Ala-Ala-Pro-Phe-pNA being the most rapidly catalyzed substrate. The cytoplasmic cyclophilin also displays a chaperone function in the citrate synthase thermal aggregation assay. Using real-time quantitative RT-PCR, we demonstrate that AvppiB is expressed under various physiological and growth conditions, mainly upregulated by acetate and downregulated by the stationary growth state, while AvppiA shows a tendency for downregulation under the tested conditions. Further, we identified chaperone protein dnaK and UDP-2, 3-diacylglucosamine hydrolase lpxH as probable interacting partners of AvPPIB and we demonstrate their physical interaction by coexpression studies. An increase in AvPPIB PPIase activity in the presence of AvdnaK and a decrease in the presence of AvlpxH further confirms each interaction. However, the PPIase activity does not seem to be essential for those interactions since AvPPIB active site mutants still interact with dnaK and lpxH, while their minor PPIase activity cannot be modulated by the interaction.     

Dissecting the Microscopic Steps of the Cyclophilin A Enzymatic Cycle on the Biological HIV-1 Capsid Substrate by NMR

Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active-site residues not only reduces the catalytic activity of these enzymes but also dramatically affects substrate binding. Employing the cyclophilin A PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the human immunodeficiency virus type 1 capsid (CA^N) protein, we demonstrate here how to dissect residue-specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of cyclophilin A active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based only on a peptide assay catalyze the human immunodeficiency virus capsid with wild-type activity but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.     

Loss of Jak2 Selectively Suppresses DC-Mediated Innate Immune Response and Protects Mice from Lethal Dose of LPS-Induced Septic Shock

Given the importance of Jak2 in cell signaling, a critical role for Jak2 in immune cells especially dendritic cells (DCs) has long been proposed. The exact function for Jak2 in DCs, however, remained poorly understood as Jak2 deficiency leads to embryonic lethality. Here we established Jak2 deficiency in adult Cre^+/+Jak2^fl/fl mice by tamoxifen induction. Loss of Jak2 significantly impaired DC development as manifested by reduced BMDC yield, smaller spleen size and reduced percentage of DCs in total splenocytes. Jak2 was also crucial for the capacity of DCs to mediate innate immune response. Jak2^−/− DCs were less potent in response to inflammatory stimuli and showed reduced capacity to secrete proinflammatory cytokines such as TNFα and IL-12. As a result, Jak2^−/− mice were defective for the early clearance of Listeria after infection. However, their potency to mediate adaptive immune response was not affected. Unlike DCs, Jak2^−/− macrophages showed similar capacity secretion of proinflammatory cytokines, suggesting that Jak2 selectively modulates innate immune response in a DC-dependent manner. Consistent with these results, Jak2^−/− mice were remarkably resistant to lethal dose of LPS-induced septic shock, a deadly sepsis characterized by the excessive innate immune response, and adoptive transfer of normal DCs restored their susceptibility to LPS-induced septic shock. Mechanistic studies revealed that Jak2/SATA5 signaling is pivotal for DC development and maturation, while the capacity for DCs secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling.     

Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis

Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.     

Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast

Cyclophilin proteins are the members of immunophillin group of proteins, known for their property of binding to the immune-suppressant drug cyclosporin A, hence named as cyclophilins. These proteins are characterized by the presence of peptidyl prolyl isomerase (PPIase) domain which catalyzes the cis-trans isomerisation process of proline residues. In the present study, an in-silico based approach was followed to identify and characterize the cyclophilin family from rice, Arabidopsis and yeast. We were able to identify 28 rice, 35 Arabidopsis and 8 yeast cyclophilin genes from their respective genomes on the basis of their annotation as well as the presence of highly conserved PPIase domain. The evolutionary relationship of the cyclophilin genes from the three genomes was analyzed using the phylogenetic tree. We have also classified the rice cyclophilin genes on the basis of localization of the protein in cell. The structural similarity of the cyclophilins was also analyzed on the basis of their homology model. The expression analysis performed using Genevestigator revealed a very strong stress responsive behavior of the gene family which was more prominent in later stages of stress. The study indicates the importance of the gene family in stress response as well as several developmental stages thus opening up many avenues for future study on the cyclophilin proteins.     

Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3

Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (K_i) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu²⁺, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca²⁺-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca²⁺ signaling and cyclophilin activity in Arabidopsis.     

Brain-expressed X-linked 2 Is Pivotal for Hyperactive Mechanistic Target of Rapamycin (mTOR)-mediated Tumorigenesis

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain-expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2^−/− mouse embryonic fibroblasts, Pten^−/− mouse embryonic fibroblasts, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal specific Tsc1 knock-out mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic specific Pten knock-out mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.     

Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex,G-Calpain,Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.     

Sex-Specific Effects on Spatial Learning and Memory,and Sex-Independent Effects on Blood Pressure of a <3.3 Mbp Rat Chromosome 2 QTL Region in Dahl Salt-Sensitive Rats

Epidemiological studies have consistently found that hypertension is associated with poor cognitive performance. We hypothesize that a putative causal mechanism underlying this association is due to genetic loci affecting both blood pressure and cognition. Consistent with this notion, we reported several blood pressure (BP) quantitative trait loci (QTLs) that co-localized with navigational performance (Nav)-QTLs influencing spatial learning and memory in Dahl rats. The present study investigates a chromosome 2 region harboring BP-f4 and Nav-8 QTLs. We developed two congenic strains, S.R2A and S.R2B introgressing Dahl R-chromosome 2 segments into Dahl S chromosome 2 region spanning BP-f4 and Nav-8 QTLs. Radiotelemetric blood pressure analysis identified only S.R2A congenic rats with lower systolic blood pressure (females: −26.0 mmHg, P = 0.003; males: −30.9 mmHg, P<1×10⁻⁵), diastolic blood pressure (females: −21.2 mmHg, P = 0.01; males: −25.7 mmHg, P<1×10⁻⁵), and mean arterial pressure (females: −23.9 mmHg, P = 0.004; males: −28.0 mmHg, P<1×10⁻⁵) compared with corresponding Dahl S controls, confirming the presence of BP-f4 QTL on rat chromosome 2. The S.R2B congenic segment did not affect blood pressure. Testing of S.R2A, S.R2B, and Dahl S male rats in the Morris water maze (MWM) task revealed significantly decreased spatial navigation performance in S.R2A male congenic rats when compared with Dahl S male controls (P<0.05). The S.R2B congenic segment did not affect performance of the MWM task in males. The S.R2A female rats did not differ in spatial navigation when compared with Dahl S female controls, indicating that the Nav-8 effect on spatial navigation is male-specific. Our results suggest the existence of a single QTL on chromosome 2 176.6–179.9 Mbp region which affects blood pressure in both males and females and cognition solely in males.     

Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Targeted Inactivation of p12Cdk2ap1, CDK2 Associating Protein 1, Leads to Early Embryonic Lethality

Targeted disruption of murine Cdk2ap1, an inhibitor of CDK2 function and hence G1/S transition, results in the embryonic lethality with a high penetration rate. Detailed timed pregnancy analysis of embryos showed that the lethality occurred between embryonic day 3.5 pc and 5.5 pc, a period of implantation and early development of implanted embryos. Two homozygous knockout mice that survived to term showed identical craniofacial defect, including a short snout and a round forehead. Examination of craniofacial morphology by measuring Snout Length (SL) vs. Face Width (FW) showed that the Cdk2ap1^+/− mice were born with a reduced SL/FW ratio compared to the Cdk2ap1^+/+ and the reduction was more pronounced in Cdk2ap1^−/− mice. A transgenic rescue of the lethality was attempted by crossing Cdk2ap1^+/− animals with K14-Cdk2ap1 transgenic mice. Resulting Cdk2ap1^{+/−:K14-Cdk2ap1} transgenic mice showed an improved incidence of full term animals (16.7% from 0.5%) on a Cdk2ap1^−/− background. Transgenic expression of Cdk2ap1 in Cdk2ap1^{−/−:K14-Cdk2ap1} animals restored SL/FW ratio to the level of Cdk2ap1^{+/−:K14-Cdk2ap1} mice, but not to that of the Cdk2ap1^{+/+:K14-Cdk2ap1} mice. Teratoma formation analysis using mESCs showed an abrogated in vivo pluripotency of Cdk2ap1^−/− mESCs towards a restricted mesoderm lineage specification. This study demonstrates that Cdk2ap1 plays an essential role in the early stage of embryogenesis and has a potential role during craniofacial morphogenesis.     

Endothelin-2-Mediated Protection of Mutant Photoreceptors in Inherited Photoreceptor Degeneration

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2^−/− mice carrying homozygous Pde6b^rd1 alleles or the Tg(RHO P347S) transgene. In the Edn2^−/− background, PR survival increased 110% in Pde6b^rd1/rd1 mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b^rd1/rd1; Edn2^−/− mice. This finding, together with systemic abnormalities in Edn2^−/− mice, suggested that the increased survival of mutant PRs in the Edn2^−/− background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b^rd1/rd1; Edn2^−/− PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b^rd1/rd1 retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2^−/− retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.