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1.  ALK-driven tumors and targeted therapy: focus on crizotinib 
Receptor tyrosine kinases have emerged as promising therapeutic targets for a diverse set of tumors. Overactivation of the tyrosine kinase anaplastic lymphoma kinase (ALK) has been reported in several types of malignancies such as anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, neuroblastoma, and non-small-cell lung carcinoma. Further characterization of the molecular role of ALK has revealed an oncogenic signaling signature that results in tumor dependence on ALK. ALK-positive tumors display a different behavior than their ALK-negative counterparts; however, the specific role of ALK in some of these tumors remains to be elucidated. Although more studies are required to establish selective targeting of ALK as a definitive therapeutic option, initial trials have shown extraordinary results in the majority of cases.
PMCID: PMC3977456  PMID: 24715763
lymphoma; therapy; kinase; pathobiology
2.  Multiprotein Complexes of Retinitis Pigmentosa GTPase Regulator (RPGR), a Ciliary Protein Mutated in X-Linked Retinitis Pigmentosa (XLRP) 
Mutations in Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of X-linked Retinitis Pigmentosa (XLRP). The RPGR gene undergoes extensive alternative splicing and encodes for distinct protein isoforms in the retina. Extensive studies using isoform-specific antibodies and mouse mutants have revealed that RPGR predominantly localizes to the transition zone to primary cilia and associates with selected ciliary and microtubule-associated assemblies in photoreceptors. In this chapter, we have summarized recent advances on understanding the role of RPGR in photoreceptor protein trafficking. We also provide new evidence that suggests the existence of discrete RPGR multiprotein complexes in photoreceptors. Piecing together the RPGR-interactome in different subcellular compartments should provide critical insights into the role of alternative RPGR isoforms in associated orphan and syndromic retinal degenerative diseases.
PMCID: PMC3464500  PMID: 20238008
3.  RPGR-containing protein complexes in syndromic and non-syndromic retinal degeneration due to ciliary dysfunction 
Journal of genetics  2009;88(4):399-407.
Dysfunction of primary cilia due to mutations in cilia-centrosomal proteins is associated with pleiotropic disorders. The primary (or sensory) cilium of photoreceptors mediates polarized trafficking of proteins for efficient phototransduction. Retinitis pigmentosa GTPase regulator (RPGR) is a cilia-centrosomal protein mutated in >70% of X-linked RP cases and 10%–20% of simplex RP males. Accumulating evidence indicates that RPGR may facilitate the orchestration of multiple ciliary protein complexes. Disruption of these complexes due to mutations in component proteins is an underlying cause of associated photoreceptor degeneration. Here, we highlight the recent developments in understanding the mechanism of cilia-dependent photoreceptor degeneration due to mutations in RPGR and RPGR-interacting proteins in severe genetic diseases, including retinitis pigmentosa, Leber congenital amaurosis (LCA), Joubert syndrome, and Senior–Loken syndrome, and explore the physiological relevance of photoreceptor ciliary protein complexes.
PMCID: PMC3464916  PMID: 20090203
primary cilia; centrosome; transition zone; ciliopathies; photoreceptor; retinal degeneration; retina; RPGR; RP2; CEP290; RPGRIP1L; NPHP
5.  Expression and Functional Roles of Caspase-5 in Inflammatory Responses of Human Retinal Pigment Epithelial Cells 
Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation of caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.
To investigate the expression, activation, and functional involvement of caspase-5 in human retinal pigment epithelial (hRPE) cells.
Expression and activation of caspase-5 in primary cultured hRPE cells, telomerase-immortalized hTERT-RPE1 cells (hTERT-RPE1), or both, were measured after stimulation with proinflammatory agents IL-1β, TNF-α, lipopolysaccharide (LPS), interferon-γ, monocyte coculture, adenosine triphosphate (ATP), or endoplasmic reticulum (ER) stress inducers. Immunomodulating agents dexamethasone (Dex), IL-10, and triamcinolone acetonide (TA) were used to antagonize proinflammatory stimulation. Cell death ELISA and TUNEL staining assays were used to assess apoptosis.
Caspase-5 mRNA expression and protein activation were induced by LPS and monocyte-hRPE coculture. Caspase-5 activation appeared as early as 2 hours after challenge by LPS and consistently increased to 24 hours. Meanwhile, caspase-1 expression and protein activation were induced by LPS. Activation of caspase-5 was blocked or reduced by Dex, IL-10, and TA. Activation of caspase-5 and -1 was also enhanced by ATP and ER stress inducers. Expression and activation of caspase-5 were inhibited by a caspase-1–specific inhibitor. Caspase-5 knockdown reduced caspase-1 protein expression and activation and inhibited TNF-α–induced IL-8 and MCP-1. In contrast to caspase-4, the contribution of caspase-5 to stress-induced apoptosis was moderate.
Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation between caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.
PMCID: PMC3230287  PMID: 21969293
6.  OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome 
Human Molecular Genetics  2012;21(15):3333-3344.
Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.
PMCID: PMC3392109  PMID: 22543976
7.  Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis  
The Journal of Clinical Investigation  2012;122(4):1233-1245.
Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.
PMCID: PMC3314468  PMID: 22446187
8.  TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein 
Human Molecular Genetics  2010;20(5):975-987.
We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.
PMCID: PMC3033188  PMID: 21159800
9.  Functional Analysis of Retinitis Pigmentosa 2 (RP2) Protein Reveals Variable Pathogenic Potential of Disease-Associated Missense Variants 
PLoS ONE  2011;6(6):e21379.
Genetic mutations are frequently associated with diverse phenotypic consequences, which limits the interpretation of the consequence of a variation in patients. Mutations in the retinitis pigmentosa 2 (RP2) gene are associated with X-linked RP, which is a phenotypically heterogenic form of retinal degeneration. The purpose of this study was to assess the functional consequence of disease-associated mutations in the RP2 gene using an in vivo assay. Morpholino-mediated depletion of rp2 in zebrafish resulted in perturbations in photoreceptor development and microphthalmia (small eye). Ultrastructural and immunofluorescence analyses revealed defective photoreceptor outer segment development and lack of expression of photoreceptor-specific proteins. The retinopathy phenotype could be rescued by expressing the wild-type human RP2 protein. Notably, the tested RP2 mutants exhibited variable degrees of rescue of rod versus cone photoreceptor development as well as microphthalmia. Our results suggest that RP2 plays a key role in photoreceptor development and maintenance in zebrafish and that the clinical heterogeneity associated with RP2 mutations may, in part, result from its potentially distinct functional relevance in rod versus cone photoreceptors.
PMCID: PMC3124502  PMID: 21738648
10.  Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy 
Nature genetics  2010;42(10):840-850.
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders featuring dysplasia or degeneration preferentially in kidney, retina, and cerebellum. Here we combine homozygosity mapping with candidate gene analysis by performing “ciliopathy candidate exome capture” followed by massively-parallel sequencing. We detect 12 different truncating mutations of SDCCAG8 in 10 NPHP-RC families. We demonstrate that SDCCAG8 is localized at both centrioles and directly interacts with NPHP-RC-associated OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in 3D renal cell cultures. This work identifies SDCCAG8 loss of function as a novel cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
PMCID: PMC2947620  PMID: 20835237
11.  Human retinopathy-associated ciliary protein retinitis pigmentosa GTPase regulator mediates cilia-dependent vertebrate development 
Human Molecular Genetics  2009;19(1):90-98.
Dysfunction of primary cilia is associated with tissue-specific or syndromic disorders. RPGR is a ciliary protein, mutations in which can lead to retinitis pigmentosa (RP), cone-rod degeneration, respiratory infections and hearing disorders. Though RPGR is implicated in ciliary transport, the pathogenicity of RPGR mutations and the mechanism of underlying phenotypic heterogeneity are still unclear. Here we have utilized genetic rescue studies in zebrafish to elucidate the effect of human disease-associated mutations on its function. We show that rpgr is expressed predominantly in the retina, brain and gut of zebrafish. In the retina, RPGR primarily localizes to the sensory cilium of photoreceptors. Antisense morpholino (MO)-mediated knockdown of rpgr function in zebrafish results in reduced length of Kupffer's vesicle (KV) cilia and is associated with ciliary anomalies including shortened body-axis, kinked tail, hydrocephaly and edema but does not affect retinal development. These phenotypes can be rescued by wild-type (WT) human RPGR. Several of the RPGR mutants can also reverse the MO-induced phenotype, suggesting their potential hypomorphic function. Notably, selected RPGR mutations observed in XLRP (T99N, E589X) or syndromic RP (T124fs, K190fs and L280fs) do not completely rescue the rpgr-MO phenotype, indicating a more deleterious effect of the mutation on the function of RPGR. We propose that RPGR is involved in cilia-dependent cascades during development in zebrafish. Our studies provide evidence for a heterogenic effect of the disease-causing mutations on the function of RPGR.
PMCID: PMC2792150  PMID: 19815619
12.  CSPP Is a Ciliary Protein Interacting with Nephrocystin 8 and Required for Cilia Formation 
Molecular Biology of the Cell  2010;21(15):2555-2567.
CSPP and CSPP-L are centrosomal proteins of known mitotic function. Here, we identify CSPP proteins as ciliary proteins and place them into a NPHP protein network crucial for normal cilia-dependent renal and retinal tissue architecture. Importantly, CSPP-L is found to be required for ciliogenesis and shown to be a cilia length modulator.
We described previously the cell cycle- and microtubule-related functions of two splice isoforms of the centrosome spindle pole-associated protein (CSPP and CSPP-L). Here, we show that endogenous CSPP isoforms not only localize to centrosomes and the midbody in cycling cells but also extend to the cilia axoneme in postmitotic resting cells. They are required for ciliogenesis in hTERT-RPE1 cells in vitro and are expressed in ciliated renal, retinal, and respiratory cells in vivo. We report that CSPP isoforms require their common C-terminal domain to interact with Nephrocystin 8 (NPHP8/RPGRIP1L) and to form a ternary complex with NPHP8 and NPHP4. We find CSPP-L to be required for the efficient localization of NPHP8 but not NPHP4 to the basal body. The ciliogenesis defect in hTERT-RPE1 cells is, however, not mediated through loss of NPHP8. Similar to the effects of ectopical expression of CSPP-L, cilia length increased in NPHP8-depleted cells. Our results thus suggest that CSPP proteins may be involved in further cytoskeletal organization of the basal body and its primary cilium. To conclude, we have identified a novel, nonmitotic function of CSPP proteins placing them into a ciliary protein network crucial for normal renal and retinal tissue architecture and physiology.
PMCID: PMC2912343  PMID: 20519441
13.  Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse 
Transgenic research  2009;19(4):691-701.
Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly expressed in the brain, peripheral sensory organs, and testis. Together, these data confirm that Supv3L1 is an important developmentally regulated gene, which continues to be expressed in all mature tissues, particularly the rapidly proliferating cells of testes, but also in the brain and sensory organs. The transgenic mice and cell lines derived from them constitute a valuable tool for the examination of the spatial and temporal aspects of Supv3L1 promoter activity, and should facilitate future screens for small molecules that regulate Supv3L1 expression.
PMCID: PMC2888911  PMID: 19937380
Supv3L1; Suv3; mouse; expression pattern; retina; EGFP reporter
14.  Interaction of retinitis pigmentosa GTPase regulator (RPGR) with RAB8A GTPase: implications for cilia dysfunction and photoreceptor degeneration 
Human Molecular Genetics  2010;19(18):3591-3598.
Defects in biogenesis or function(s) of primary cilia are associated with numerous inherited disorders (called ciliopathies) that may include retinal degeneration phenotype. The cilia-expressed gene RPGR (retinitis pigmentosa GTPase regulator) is mutated in patients with X-linked retinitis pigmentosa (XLRP) and encodes multiple protein isoforms with a common N-terminal domain homologous to regulator of chromosome condensation 1 (RCC1), a guanine nucleotide exchange factor (GEF) for Ran GTPase. RPGR interacts with several ciliopathy proteins, such as RPGRIP1L and CEP290; however, its physiological role in cilia-associated functions has not been delineated. Here, we report that RPGR interacts with the small GTPase RAB8A, which participates in cilia biogenesis and maintenance. We show that RPGR primarily associates with the GDP-bound form of RAB8A and stimulates GDP/GTP nucleotide exchange. Disease-causing mutations in RPGR diminish its interaction with RAB8A and reduce the GEF activity. Depletion of RPGR in hTERT-RPE1 cells interferes with ciliary localization of RAB8A and results in shorter primary cilia. Our data suggest that RPGR modulates intracellular localization and function of RAB8A. We propose that perturbation of RPGR–RAB8A interaction, at least in part, underlies the pathogenesis of photoreceptor degeneration in XLRP caused by RPGR mutations.
PMCID: PMC2928130  PMID: 20631154
16.  Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy  
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
PMCID: PMC2827951  PMID: 20179356
17.  Interaction of ciliary disease protein retinitis pigmentosa GTPase regulator with nephronophthisis-associated proteins in mammalian retinas 
Molecular Vision  2010;16:1373-1381.
Retinitis pigmentosa GTPase regulator (RPGR) is a cilia-centrosomal protein that frequently mutates in X-linked retinal degeneration and associated disorders. RPGR interacts with multiple ciliary proteins in the retina. Perturbations in the assembly of RPGR complexes are associated with retinal degeneration. This study was undertaken to delineate the composition and dissection of RPGR complexes in mammalian retinas.
Immunoprecipitation of RPGR from ciliary fraction of bovine retina was performed, followed by mass spectrometry analysis. The glutathione S-transferase pull-down assay was performed to validate the interaction. Immunodepletion experiments were performed to dissect the partitioning of RPGR in different protein complexes in mammalian retinas.
We found that RPGR associates with a ciliary protein nephrocystin-4 (nephroretinin; NPHP4) that is mutated in nephronophthisis (NPH) and RP (Senior-Løken syndrome). This association is abolished in the Rpgr-knockout mouse retina. The RCC1-like domain of RPGR interacts with the N-terminal 316 amino acids of NPHP4. In the retina, RPGR also associates with NPHP1, an NPHP4-interacting protein; RPGR interacts directly with amino acids 243–586 of NPHP1. We further show that, in the retina, RPGR associates with and is partitioned in at least two different complexes with NPHP-associated proteins, (i) NPHP1, NPHP2, and NPHP5, and (ii) NPHP4, NPHP6, and NPHP8.
RPGR may regulate some complexes with NPHP proteins in the mammalian retina. The disruption of these complexes may contribute to the pathogenesis of retinal degeneration in X-linked RP and associated ciliary diseases.
PMCID: PMC2905641  PMID: 20664800
18.  A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies 
Nature genetics  2009;41(6):739-745.
Despite rapid advances in disease gene identification, the predictive power of the genotype remains limited, in part due to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in patients with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss of function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the 229T-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.
PMCID: PMC2783476  PMID: 19430481
19.  Three novel polymorphic microsatellite markers for the glaucoma locus GLC1B by datamining tetranucleotide repeats on chromosome 2p12-q12 
Genetics and Molecular Biology  2009;32(4):720-722.
In order to identify new markers around the glaucoma locus GLC1B as a tool to refine its critical region at 2p11.2-2q11.2, we searched the critical region sequence obtained from the UCSC database for tetranucleotide (GATA)n and (GTCT)n repeats of at least 10 units in length. Three out of four potential microsatellite loci were found to be polymorphic, heterozygosity ranging from 64.56% to 79.59%. The identified markers are useful not only for GLC1B locus but also for the study of other disease loci at 2p11.2-2q11.2, a region with scarcity of microsatellite markers.
PMCID: PMC3036884  PMID: 21637444
GLC1B; microsatellite polymorphic markers; tetranucleotide tandem repeat; gene mapping; glaucoma

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