Gabe is an MD/PhD student at the University of Iowa who joined the Mahajan lab in 2016. Gabe received his bachelor’s degree in molecular biology from Winona State University in 2014. Gabe is studying the structure of the calpain-5 (CAPN5) protein and its role in the development of Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV), a rare blinding eye disease. His research interests include translational proteomics, structural biology, biophysical chemistry, enzymology, drug design, molecular modeling, and bioinformatics. He was awarded an NIH F30 grant in 2017 and defended his Ph.D. in 2020.
Current Role at Stanford
Graduate Research Assistant
Honors & Awards
Koichi Suzuki Predoctoral Award, The Biology of Calpains in Health and Disease Conference, Federation of American Societies for Experimental Biology (FASEB) (2019)
X-Ray Methods in Structural Biology Course Attendee, Cold Spring Harbor Laboratory (CSHL) (2019)
RapiData 2019 Macromolecular X-Ray Diffraction and Measurement Course, Stanford Synchrotron Radiation Lightsource (SSRL) (2019)
SRC Travel Award, The Biology of Calpains in Health and Disease Conference, Federation of American Societies for Experimental Biology (FASEB) (2019)
NEI Recognized Hispanic/Latino Researchers and Eye Care Professionals, National Eye Health Education Program (NEHEP) (2018)
Trainee Scholar Travel Award, University of Iowa Carver College of Medicine (2018)
Iowa Graduate Success Fellowship, University of Iowa Graduate College (2017)
Ruth L. Kirschstein National Research Service (NRSA) F30 Award, National Eye Institute (NEI) (2017)
SRC Travel Award, Biology & Chemistry of Vision Conference, Federation of American Societies for Experimental Biology (FASEB) (2017)
Arthur A. Spector Award in Basic Science Research, University of Iowa Carver College of Medicine (2014)
Medical Scientist Training Program (MSTP) Fellowship, University of Iowa Carver College of Medicine (2014)
Sloan Pre-Doctoral Scholarship, Alfred P. Sloan Foundation (2014)
Summa Cum Laude, Bachelor of Science, Winona State University (2014)
Thomas Pietsch Scholarship, Winona State University (2013)
Undergraduate Research & Creative Project Grant, Winona State University (2013)
Mayo Summer Undergraduate Research (SURF) Fellowship, Mayo Clinic Foundation (2012-2013)
Annual Dean's List, Winona State University (2010-2014)
Presidential Honor's Scholarship, Winona State University (2010-2014)
Education & Certifications
M.D., University of Iowa Carver College of Medicine, Medical Scientist Training Program (MSTP) (2022)
Ph.D., University of Iowa Carver College of Medicine, Molecular Medicine (2020)
B.S., Winona State University, Cell & Molecular Biology, Biochemistry (Minor) (2014)
C.N.A., Normandale Community College, Certified Nursing Assistant Certification (2010)
Structure and Function of Calpain-5 (CAPN5), Stanford University (June 2016 - Present)
The eye is an immune-privileged site that is particularly prone to autoinflammatory uveitis. Most causes of uveitis are unknown, thereby delaying treatment and allowing ocular inflammation to progress unabated. Mutations in the cysteine protease calpain-5 (CAPN5) cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV), a rare form of nonsyndromic uveitis. ADNIV patients develop ocular inflammation, retinal degeneration and neovascularization, and intraocular fibrosis starting in the second decade of life, culminating in blindness in their fifties. ADNIV mutations lead to the production of an overactive protease. To date, there are no calpain inhibitors specific to calpain-5 and its structure and function are poorly understood. Determining the structure of calpain-5 is critical to developing precise therapies for ADNIV. The Mahajan lab utilizes a variety of structural, enzymatic, and proteomics techniques to uncover the structure and regulatory mechanisms of this poorly understood molecule. This work is supported by an F30 training grant from the National Eye Institute (1F30EYE027986-01A1)
Palo Alto, CA
Personalized Proteomics of Liquid Biopsies, Stanford University (11/1/2014 - Present)
Proteomics is the culmination of advances in chemistry, physics, bioinformatics, and molecular biology that allow the study of thousands of proteins simultaneously. A proteome can be defined as the complete set of proteins expressed in a particular cell or tissue of interest. This strategy is especially important in eye diseases where there are limited cell and animal models. We are using proteomics to discover biomarkers, therapeutic targets, and molecular pathways involved in several eye diseases where diagnosis is difficult and current treatment is inadequate.
Palo Alto, CA
Computational Structural Modeling, Stanford University (6/1/2015 - Present)
With the rapidly-reducing costs of whole genome and exome sequencing, clinical genomic testing is being increasingly applied in the identification of pathogenic disease variants in patients. New sequence variants are often identified, but many are classified as variants of uncertain significance (VUS) due to the lack of functional assays to study their effects. Computational structural modeling of protein structures can provide information at the atomic level and help to predict the pathogenicity of novel mutations by placing variants of uncertain significance in the context of the patient’s disease, pathophysiology, and protein function. Our team collaborates with clinicians and researchers around the world to model novel disease variants and better understand their patients’ disease.
Palo Alto, CA
Service, Volunteer and Community Work
Medical Student Officer and Volunteer Examiner, Iowa City Free Eye Clinic (2015 - 2017)
Iowa City, IA
Undergraduate Research Assistant, Mayo Clinic (5/2012 - 4/2014)
Department of Surgery and Biophysics
Graduate Research Assistant, University of Iowa (6/2014 - 5/2017)
Iowa City, IA
Visiting Student Researcher, Stanford University (7/2017 - Present)
Palo Alto, CA
Professional Affiliations and Activities
Course Liaison Committee, University of Iowa Carver College of Medicine (2016 - 2016)
- Liquid biopsy proteomics of uveal melanoma reveals biomarkers associated with metastatic risk. Molecular cancer 2021; 20 (1): 39
Structure-based phylogeny identifies Avoralstat as a TMPRSS2 inhibitor that prevents SARS-CoV-2 infection in mice.
The Journal of clinical investigation
Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.
View details for DOI 10.1172/JCI147973
View details for PubMedID 33844653
Molecular Surgery: Proteomics of a Rare Genetic Disease Gives Insight into Common Causes of Blindness.
2020; 23 (11): 101667
Rare diseases are an emerging global health priority. Although individually rare, the prevalence of rare "orphan" diseases is high, affecting approximately 300 million people worldwide. Treatments for these conditions are often inadequate, leaving the disease to progress unabated. Here, we review the clinical features and pathophysiology of neovascular inflammatory vitreoretinopathy (NIV), a rare inflammatory retinal disease caused by mutations in the CAPN5 gene. Although the prevalence of NIV is low (1 in 1,000,000 people), the disease mimics more common causes of blindness (e.g. uveitis, retinitis pigmentosa, proliferative diabetic retinopathy, and proliferative vitreoretinopathy) at distinct clinical stages. There is no cure for NIV to date. We highlight how personalized proteomics helped identify potential stage-specific biomarkers and drug targets in liquid vitreous biopsies. The NIV vitreous proteome revealed enrichment of molecular pathways associated with common retinal pathologies and implicated superior targets for therapeutic drug repositioning. In addition, we review our pipeline for collecting, storing, and analyzing ophthalmic surgical samples. This approach can be adapted to treat a variety of rare genetic diseases.
View details for DOI 10.1016/j.isci.2020.101667
View details for PubMedID 33134897
Structural Insights into the Unique Activation Mechanisms of a Non-classical Calpain and Its Disease-Causing Variants.
2020; 30 (3): 881
Increased calpain activity is linked to neuroinflammation including a heritable retinal disease caused by hyper-activating mutations in the calcium-activated calpain-5 (CAPN5) protease. Although structures for classical calpains are known, the structure of CAPN5, a non-classical calpain, remains undetermined. Here we report the 2.8A crystal structure of the human CAPN5 protease core (CAPN5-PC). Compared to classical calpains, CAPN5-PC requires high calcium concentrations for maximal activity. Structure-based phylogenetic analysis and multiple sequence alignment reveal that CAPN5-PC contains three elongated flexible loops compared to its classical counterparts. The presence of a disease-causing mutation (c.799G>A, p.Gly267Ser) on the unique PC2L2 loop reveals a function in this region for regulating enzymatic activity. This mechanism could be transferred to distant calpains, using synthetic calpain hybrids, suggesting an evolutionary mechanism for fine-tuning calpain function by modifying flexible loops. Further, the open (inactive) conformation of CAPN5-PC provides structural insight into CAPN5-specific residues that can guide inhibitor design.
View details for DOI 10.1016/j.celrep.2019.12.077
View details for PubMedID 31968260
Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration.
2020; 52: 102636
Neurodegenerative diseases are incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in photoreceptor death and progresses to the loss of the entire retinal network. We previously found that proteomic analysis of the adjacent vitreous served as way to indirectly biopsy the retina and identify changes in the retinal proteome.We analyzed protein expression in liquid vitreous biopsies from autosomal recessive (ar)RP patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle.Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on ɑ-ketoglutarate-treated mice revealed restoration of metabolites that correlated with our proteomic findings: uridine, dihydrouridine, and thymidine (pyrimidine and purine metabolism), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle).This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs retinal function and provides a neuroprotective effect on the photoreceptor cells and inner retinal network.NIH grants [R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, P30EY026877, 5P30EY019007, R01EY018213, F30EYE027986, T32GM007337, 5P30CA013696], NSF grant CHE-1734082.
View details for DOI 10.1016/j.ebiom.2020.102636
View details for PubMedID 32028070
Personalized Proteomics for Precision Health: Identifying Biomarkers of Vitreoretinal Disease.
Translational vision science & technology
2018; 7 (5): 12
Proteomic analysis is an attractive and powerful tool for characterizing the molecular profiles of diseased tissues, such as the vitreous. The complexity of data available for analysis ranges from single (e.g., enzyme-linked immunosorbent assay [ELISA]) to thousands (e.g., mass spectrometry) of proteins, and unlike genomic analysis, which is limited to denoting risk, proteomic methods take snapshots of a diseased vitreous to evaluate ongoing molecular processes in real time. The proteome of diseased ocular tissues was recently characterized, uncovering numerous biomarkers for vitreoretinal diseases and identifying protein targets for approved drugs, allowing for drug repositioning. These biomarkers merit more attention regarding their therapeutic potential and prospective validation, as well as their value as reproducible, sensitive, and specific diagnostic markers.Translational Relevance: Personalized proteomics offers many advantages over alternative precision-health platforms for the diagnosis and treatment of vitreoretinal diseases, including identification of molecular constituents in the diseased tissue that can be targeted by available drugs.
View details for PubMedID 30271679
A novel de novo CAPN5 mutation in a patient with inflammatory vitreoretinopathy, hearing loss, and developmental delay.
Cold Spring Harbor molecular case studies
2018; 4 (3)
Mutations that activate the protease calpain-5 (CAPN5) cause a nonsyndromic adult-onset autoinflammatory eye disease characterized by uveitis, altered synaptic signaling, retinal degeneration, neovascularization, and intraocular fibrosis. We describe a pediatric patient with severe inflammatory vitreoretinopathy accompanied by hearing loss and developmental delay associated with a novel, de novo CAPN5 missense mutation (c.865C>T, p.Arg289Trp) that shows greater hyperactivation of the calpain protease, indicating a genotype-phenotype correlation that links mutation severity to proteolytic activity and the possibility of earlier onset syndromic disease with auditory and neurological abnormalities.
View details for PubMedID 29472286
Proteomic analysis of the human retina reveals region-specific susceptibilities to metabolic-and oxidative stress-related diseases
2018; 13 (2): e0193250
Differences in regional protein expression within the human retina may explain molecular predisposition of specific regions to ophthalmic diseases like age-related macular degeneration, cystoid macular edema, retinitis pigmentosa, and diabetic retinopathy. To quantify protein levels in the human retina and identify patterns of differentially-expressed proteins, we collected foveomacular, juxta-macular, and peripheral retina punch biopsies from healthy donor eyes and analyzed protein content by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein expression was analyzed with 1-way ANOVA, gene ontology, pathway representation, and network analysis. We identified a mean of 1,974 proteins in the foveomacular retina, 1,999 in the juxta-macular retina, and 1,779 in the peripheral retina. Six hundred ninety-seven differentially-expressed proteins included those unique to and abundant in each anatomic region. Proteins with higher expression in each region include: heat-shock protein 90-alpha (HSP90AA1), and pyruvate kinase (PKM) in the foveomacular retina; vimentin (VIM) and fructose-bisphosphate aldolase C (ALDOC); and guanine nucleotide-binding protein subunit beta-1 (GNB1) and guanine nucleotide-binding protein subunit alpha-1 (GNAT1) in the peripheral retina. Pathway analysis identified downstream mediators of the integrin signaling pathway to be highly represented in the foveomacular region (P = 6.48 e-06). Metabolic pathways were differentially expressed among all retinal regions. Gene ontology analysis showed that proteins related to antioxidant activity were higher in the juxta-macular and the peripheral retina, but present in lower amounts in the foveomacular retina. Our proteomic analysis suggests that certain retinal regions are susceptible to different forms of metabolic and oxidative stress. The findings give mechanistic insight into retina function, reveal important molecular processes, and prioritize new pathways for therapeutic targeting.
View details for PubMedID 29466423
Therapeutic drug repositioning using personalized proteomics of liquid biopsies.
2017; 2 (24)
BACKGROUND: In patients with limited response to conventional therapeutics, repositioning of already approved drugs can bring new, more effective options. Current drug repositioning methods, however, frequently rely on retrospective computational analyses and genetic testing - time consuming methods that delay application of repositioned drugs. Here, we show how proteomic analysis of liquid biopsies successfully guided treatment of neovascular inflammatory vitreoretinopathy (NIV), an inherited autoinflammatory disease with otherwise poor clinical outcomes.METHODS: Vitreous biopsies from NIV patients were profiled by an antibody array for expression of 200 cytokine-signaling proteins. Non-NIV controls were compared with NIV samples from various stages of disease progression. Patterns were identified by 1-way ANOVA, hierarchical clustering, and pathway analysis. Subjects treated with repositioned therapies were followed longitudinally.RESULTS: Proteomic profiles revealed molecular pathways in NIV pathologies and implicated superior and inferior targets for therapy. Anti-VEGF injections resolved vitreous hemorrhages without the need for vitrectomy surgery. Methotrexate injections reversed inflammatory cell reactions without the side effects of corticosteroids. Anti-IL-6 therapy prevented recurrent fibrosis and retinal detachment where all prior antiinflammatory interventions had failed. The cytokine array also showed that TNF-alpha levels were normal and that corticosteroid-sensitive pathways were absent in fibrotic NIV, helping explain prior failure of these conventional therapeutic approaches.CONCLUSIONS: Personalized proteomics can uncover highly personalized therapies for autoinflammatory disease that can be timed with specific pathologic activities. This precision medicine strategy can also help prevent delivery of ineffective drugs. Importantly, proteomic profiling of liquid biopsies offers an endpoint analysis that can directly guide treatment using available drugs.
View details for PubMedID 29263305
Proteomic analysis of autoimmune retinopathy implicates NrCAM as a potential biomarker.
2022; 2 (2)
To identify vitreous molecular biomarkers associated with autoimmune retinopathy (AIR).Case-control study.We analyzed six eyes from four patients diagnosed with AIR and eight comparative controls diagnosed with idiopathic macular holes and epiretinal membranes.Vitreous biopsies were collected from the participants and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or multiplex ELISA.Protein expression changes were evaluated by 1-way ANOVA (significant p-value <0.05), hierarchical clustering, and pathway analysis to identify candidate protein biomarkers.There were 16 significantly upregulated and 17 significantly downregulated proteins in the vitreous of three AIR patients compared to controls. The most significantly upregulated proteins included lysozyme C (LYSC), zinc-alpha-2-glycoprotein (ZA2G), complement factor D (CFAD), transforming growth factor-beta induced protein (BGH3), beta-crystallin B2, and alpha-crystallin A chain. The most significantly downregulated proteins included disco-interacting protein 2 homolog (DIP2C), retbindin (RTBDN), and amyloid beta precursor like protein 2 (APLP2). Pathway analysis revealed that vascular endothelial growth factor (VEGF) signaling was a top represented pathway in the vitreous of AIR patients compared to controls. In comparison to a different cohort of three AIR patients analyzed by multiplex ELISA, a commonly differentially expressed protein was neuronal cell adhesion molecule (NrCAM) with p-values of 0.027 in the LC-MS/MS dataset and 0.035 in the ELISA dataset.Protein biomarkers such as NrCAM in the vitreous may eventually help diagnose AIR.
View details for DOI 10.1016/j.xops.2022.100131
View details for PubMedID 35529077
View details for PubMedCentralID PMC9075676
Surgical management of acanthamoeba chorioretinitis.
American journal of ophthalmology case reports
2022; 25: 101388
Purpose: Acanthamoeba chorioretinitis is a rare manifestation of the parasitic infection, and reported cases often result in enucleation. Surgical removal of Acanthamoeba chorioretinitis has not been previously described. We report a surgical case of Acanthamoeba chorioretinitis spread from keratitis that ultimately resulted in a disease-free outcome.Observations: A healthy 80-year-old male with a history of keratoconus requiring a penetrating keratoplasty in the fellow eye presented with a severe corneal ulcer clinically consistent with Acanthamoeba keratitis. He ultimately required a penetrating keratoplasty and improved clinically until he developed vitritis on post-operative month 1 and was diagnosed with endophthalmitis. B-scan ultrasound demonstrated vitreous opacities and a large retinal mass that reduced in size following serial intravitreal injections of antibiotics, oral antibiotics, and a limited pars plana vitrectomy. He underwent a repeat pars plana vitrectomy 6 weeks later and a retinal mass in the mid-periphery with an associated tractional retinal detachment was noted. A localized retinectomy was performed around the lesion which was excised entirely, and silicone oil was instilled. Pathology of the lesion showed acute and chronic granulomatous necrotizing inflammation with the presence of several definitive amoebic organisms and numerous cells suspicious for amoebae. The patient was maintained on oral antibiotics by the Infectious Disease Service and was disease-free 1-year post-infection.Conclusions and importance: Acanthamoeba chorioretinitis is a rare, devastating disease and often leads to enucleation. We present a surgical case showing control of the infection utilizing a surgical retinectomy. Aggressive local therapy and a multidisciplinary approach with the Infectious Disease Service may lead to a successful outcome.
View details for DOI 10.1016/j.ajoc.2022.101388
View details for PubMedID 35243145
Proteomic identification of candidate biomarkers that distinguish lens-induced uveitis from infectious endophthalmitis
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2021
View details for Web of Science ID 000690761400154
Peptidomimetics Therapeutics for Retinal Disease.
2021; 11 (3)
Ocular disorders originating in the retina can result in a partial or total loss of vision, making drug delivery to the retina of vital importance. However, effectively delivering drugs to the retina remains a challenge for ophthalmologists due to various anatomical and physicochemical barriers in the eye. This review introduces diverse administration routes and the accordant pharmacokinetic profiles of ocular drugs to aid in the development of safe and efficient drug delivery systems to the retina with a focus on peptidomimetics as a growing class of retinal drugs, which have great therapeutic potential and a high degree of specificity. We also discuss the pharmacokinetic profiles of small molecule drugs due to their structural similarity to small peptidomimetics. Lastly, various formulation strategies are suggested to overcome pharmacokinetic hurdles such as solubility, retention time, enzymatic degradation, tissue targeting, and membrane permeability. This knowledge can be used to help design ocular delivery platforms for peptidomimetics, not only for the treatment of various retinal diseases, but also for the selection of potential peptidomimetic drug targets.
View details for DOI 10.3390/biom11030339
View details for PubMedID 33668179
Whole-Exome Sequencing of Patients with Posterior Segment Uveitis.
American journal of ophthalmology
OBJECTIVE: To elucidate molecular risk factors for posterior segment uveitis using a functional genomics approach.DESIGN: Genetic Association Cohort Study.METHODS: SETTING: Single-center study at an academic referral center.STUDY POPULATION: 164 patients with clinically diagnosed uveitis of the posterior segment.MAIN OUTCOME MEASURES: Exome sequencing was used to detect variants identified in 164 patients with posterior segment uveitis. A phenotype-driven analysis, protein structural modeling and in silico calculations were then used to rank and predict the functional consequences of key variants.RESULTS: A total of 203 single nucleotide variants, in 23 genes across 164 patients, were included in this study. Both known and novel variants were identified in genes previously implicated in specific types of syndromic uveitis - such as NOD2 (Blau Syndrome) and CAPN5 NIV (Neovascular Inflammatory Vitreoretinopathy) - as well as variants in genes not previously linked to posterior segment uveitis. Based on a ranked list and protein-protein-interaction network, missense variants in NOD-like receptor family genes (NOD2, NLRC4, NLRP3, and NLRP1), CAPN5, and TYK2 were characterized via structural modeling and in silico calculations to predict how specific variants might alter protein structure and function. The majority of analyzed variants were notably different from wild type.CONCLUSIONS: This study implicates new pathways and immune signaling proteins that may be associated with posterior segment uveitis susceptibility. A larger cohort and functional studies will help validate the pathogenicity of the mutations identified. In specific cases, whole exome sequencing can help diagnose non-syndromic uveitis patients harboring known variants for syndromic inflammatory diseases.
View details for DOI 10.1016/j.ajo.2020.07.021
View details for PubMedID 32707200
Proteomic analysis of intermediate uveitis suggests myeloid cell recruitment and implicates IL-23 as a therapeutic target.
American journal of ophthalmology case reports
2020; 18: 100646
To profile vitreous protein expression of intermediate uveitis (IU) patients.We identified a mean of 363 ± 41 unique proteins (mean ± SD) in IU vitreous and 393 ± 69 unique proteins in control samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of liquid vitreous biopsies collected during pars plana vitrectomy. A total of 233 proteins were differentially expressed among control and IU samples, suggesting a protein signature that could distinguish the two groups. Pathway analysis identified 22 inflammatory mediators of the interleukin-12 (IL-12) signaling pathway in IU vitreous. Upstream regulator analysis identified downstream mediators of IL-23 and myeloid differentiation primary response protein (MYD88), both of which are involved in the recruitment and differentiation of myeloid cells. Taken together, our results suggest the recruitment of myeloid cells as an upstream pathway in the pathogenesis of IU.This study provides insights into proteins that will serve as biomarkers and therapeutic targets for IU. These biomarkers will help design future clinical trials using rational molecular therapeutics.
View details for DOI 10.1016/j.ajoc.2020.100646
View details for PubMedID 32274442
View details for PubMedCentralID PMC7132169
- Mechanisms of neurodegeneration in a preclinical autosomal dominant retinitis pigmentosa knock-in model with a Rho(D190N) mutation CELLULAR AND MOLECULAR LIFE SCIENCES 2019; 76 (18): 3657–65
CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials.
Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 knockout mouse model. In humans, CAPN5 loss-of-function gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 knockout mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 loss-of-function CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these loss-of-function variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the loss-of-function of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/humu.23894
View details for PubMedID 31403230
CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
View details for Web of Science ID 000488628100371
Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans.
Proceedings of the National Academy of Sciences of the United States of America
Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr Ali18 are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.
View details for DOI 10.1073/pnas.1819825116
View details for PubMedID 31138708
- SCAPER-associated nonsyndromic autosomal recessive retinitis pigmentosa AMERICAN JOURNAL OF MEDICAL GENETICS PART A 2019; 179 (2): 312–16
- VCAN Canonical Splice Site Mutation is Associated With Vitreoretinal Degeneration and Disrupts an MMP Proteolytic Site INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 2019; 60 (1): 282–93
Novel REEP6 gene mutation associated with autosomal recessive retinitis pigmentosa.
Documenta ophthalmologica. Advances in ophthalmology
This study reports the ophthalmic and genetic findings of a Cameroonian patient with autosomal recessive retinitis pigmentosa (arRP) caused by a novel Receptor Expression Enhancing Protein 6 (REEP6) homozygous mutation.A 33-year-old man underwent comprehensive ophthalmic examinations, including visual acuity measurements, dilated fundus imaging, electroretinography (ERG), and spectral-domain optical coherence tomography (SD-OCT). Short-wavelength fundus autofluorescence (SW-AF) and near-infrared fundus autofluorescence (NIR-AF) were also evaluated. Whole exome sequencing (WES) was used to identify potential pathogenic variants.Fundus examination revealed typical RP findings with additional temporal ten micron yellow dots. SD-OCT imaging revealed cystoid macular edema and perifoveal outer retinal atrophy with centrally preserved inner segment ellipsoid zone (EZ) bands. Hyperreflective spots were seen in the inner retinal layers. On SW-AF images, a hypoautofluorescent area in the perifoveal area was observed. NIR-AF imaging revealed an irregularly shaped hyperautofluorescent ring. His visual acuity was mildly affected. ERG showed undetectable rod responses and intact cone responses. Genetic testing via WES revealed a novel homozygous mutation (c.295G>A, p.Glu99Lys) in the gene encoding REEP6, which is predicted to alter the charge in the transmembrane helix.This report is not only the first description of a Cameroonian patient with arRP associated with a REEP6 mutation, but also this particular genetic alteration. Substitution of p.Glu99Lys in REEP6 likely disrupts the interactions between REEP6 and the ER membrane. NIR-AF imaging may be particularly useful for assessing functional photoreceptor cells and show an "avocado" pattern of hyperautofluorescence in patients with the REEP6 mutation.
View details for DOI 10.1007/s10633-019-09719-1
View details for PubMedID 31538292
VCAN Canonical Splice Site Mutation is Associated With Vitreoretinal Degeneration and Disrupts an MMP Proteolytic Site.
Investigative ophthalmology & visual science
2019; 60 (1): 282–93
To gain insight into the pathophysiology of vitreoretinal degeneration, the clinical course of three family members with Versican Vitreoretinopathy (VVR) is described, and a canonical splice site mutation in the gene encoding for versican (VCAN) protein was biochemically analyzed.A retrospective chart review, human eye histopathology, Sanger DNA sequencing, protein structural modeling, and in vitro proteolysis assays were performed.The proband (II:1), mother (I:2), and younger sibling (II:2) suffered retinal degeneration with foveal sparing and retinal detachments with proliferative vitreoretinopathy, features that were confirmed on histopathologic analysis. All affected members carried a heterozygous adenine to guanine variant (c.4004-2A>G) predicted to result in exon 8 skipping or the deletion of 13 amino acids at the beginning of the GAGβ chain (VCAN p.1335-1347). This deleted region corresponded to a putative MMP cleavage site, validated using fluorescence resonance energy transfer (FRET)-based proteolysis assays. Proteomic network analysis identified 10 interacting partners in the human vitreous and retina linked to retinal detachment and degeneration.VVR causes significant ocular disease, including retinal detachment and retinal dystrophy. The intronic VCAN mutation removes an MMP cleavage site, which alters versican structure and results in abnormal vitreous modeling. Disruption of a versican protein network may underlie clinicopathologic disease features and point to targeted therapies.
View details for PubMedID 30657523
In trans variant calling reveals enrichment for compound heterozygous variants in genes involved in neuronal development and growth.
2019; 101: e8
Compound heterozygotes occur when different variants at the same locus on both maternal and paternal chromosomes produce a recessive trait. Here we present the tool VarCount for the quantification of variants at the individual level. We used VarCount to characterize compound heterozygous coding variants in patients with epileptic encephalopathy and in the 1000 Genomes Project participants. The Epi4k data contains variants identified by whole exome sequencing in patients with either Lennox-Gastaut Syndrome (LGS) or infantile spasms (IS), as well as their parents. We queried the Epi4k dataset (264 trios) and the phased 1000 Genomes Project data (2504 participants) for recessive variants. To assess enrichment, transcript counts were compared between the Epi4k and 1000 Genomes Project participants using minor allele frequency (MAF) cutoffs of 0.5 and 1.0%, and including all ancestries or only probands of European ancestry. In the Epi4k participants, we found enrichment for rare, compound heterozygous variants in six genes, including three involved in neuronal growth and development - PRTG (p = 0.00086, 1% MAF, combined ancestries), TNC (p = 0.022, 1% MAF, combined ancestries) and MACF1 (p = 0.0245, 0.5% MAF, EU ancestry). Due to the total number of transcripts considered in these analyses, the enrichment detected was not significant after correction for multiple testing and higher powered or prospective studies are necessary to validate the candidacy of these genes. However, PRTG, TNC and MACF1 are potential novel recessive epilepsy genes and our results highlight that compound heterozygous variants should be considered in sporadic epilepsy.
View details for DOI 10.1017/S0016672319000065
View details for PubMedID 31190668
Proteomic insight into the pathogenesis of CAPN5-vitreoretinopathy.
2019; 9 (1): 7608
CAPN5 Neovascular Inflammatory Vitreoretinopathy (CAPN5-NIV; OMIM 193235) is a poorly-understood rare, progressive inflammatory intraocular disease with limited therapeutic options. To profile disease effector proteins in CAPN5-NIV patient vitreous, liquid vitreous biopsies were collected from two groups: eyes from control subjects (n = 4) with idiopathic macular holes (IMH) and eyes from test subjects (n = 12) with different stages of CAPN5-NIV. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein expression changes were evaluated by principal component analysis, 1-way ANOVA (significant p-value < 0.05), hierarchical clustering, gene ontology, and pathway representation. There were 216 differentially-expressed proteins (between CAPN5-NIV and control vitreous), including those unique to and abundant in each clinical stage. Gene ontology analysis revealed decreased synaptic signaling proteins in CAPN5-NIV vitreous compared to controls. Pathway analysis revealed that inflammatory mediators of the acute phase response and the complement cascade were highly-represented. The CAPN5-NIV vitreous proteome displayed characteristic enrichment of proteins and pathways previously-associated with non-infectious posterior uveitis, rhegmatogenous retinal detachment (RRD), age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR). This study expands our knowledge of affected molecular pathways in CAPN5-NIV using unbiased, shotgun proteomic analysis rather than targeted detection platforms. The high-levels and representation of acute phase response proteins suggests a functional role for the innate immune system in CAPN5-NIV pathogenesis.
View details for DOI 10.1038/s41598-019-44031-7
View details for PubMedID 31110225
SCAPER-associated nonsyndromic autosomal recessive retinitis pigmentosa.
American journal of medical genetics. Part A
Mutations in the gene SCAPER (S-phase CyclinA Associated Protein residing in the Endoplasmic Reticulum) have recently been identified as causing syndromic autosomal recessive retinitis pigmentosa with the extraocular manifestations of intellectual disability and attention-deficit/hyperactivity disorder. We present the case of an 11-year-old boy that presented to our clinic with the complaint of decreased night vision. Clinical presentation, family history, and diagnostic imaging were congruent with the diagnosis of autosomal recessive retinitis pigmentosa. Genetic testing of the patient and both parents via whole-exome sequencing revealed the homozygous mutation c.2023-2A>G in SCAPER. Unique to our patient's presentation is the absence of intellectual disability and attention-deficit/hyperactivity disorder, suggesting that SCAPER-associated retinitis pigmentosa can also present without systemic manifestations.
View details for PubMedID 30561111
ProSave: an application for restoring quantitative data to manipulated subsets of protein lists.
Source code for biology and medicine
2018; 13: 3
In proteomics studies, liquid chromatography tandem mass spectrometry data (LC-MS/MS) is quantified by spectral counts or by some measure of ion abundance. Downstream comparative analysis of protein content (e.g. Venn diagrams and network analysis) typically does not include this quantitative data and critical information is often lost. To avoid loss of spectral count data in comparative proteomic analyses, it is critical to implement a tool that can rapidly retrieve this information.We developed ProSave, a free and user-friendly Java-based program that retrieves spectral count data from a curated list of proteins in a large proteomics dataset. ProSave allows for the management of LC-MS/MS datasets and rapidly retrieves spectral count information for a desired list of proteins.ProSave is open source and freely available at https://github.com/MahajanLab/ProSave. The user manual, implementation notes, and description of methodology and examples are available on the site.
View details for DOI 10.1186/s13029-018-0070-0
View details for PubMedID 30459825
View details for PubMedCentralID PMC6233572
- ProSave: an application for restoring quantitative data to manipulated subsets of protein lists SOURCE CODE FOR BIOLOGY AND MEDICINE 2018; 13
- Personalized Proteomics for Precision Health: Identifying Biomarkers of Vitreoretinal Disease TRANSLATIONAL VISION SCIENCE & TECHNOLOGY 2018; 7 (5)
- Extracellular superoxide dismutase (SOD3) regulates oxidative stress at the vitreoretinal interface FREE RADICAL BIOLOGY AND MEDICINE 2018; 124: 408–19
Missense mutation in SLIT2 associated with congenital myopia, anisometropia, connective tissue abnormalities, and obesity
ORPHANET JOURNAL OF RARE DISEASES
2018; 13: 138
SLIT2 is a protein ligand for the Roundabout (ROBO) receptor and was found to play a major role in repulsive midline axon guidance in central nervous system development. Based on studies utilizing knockout models, it has been postulated that SLIT2 is important for preventing inappropriate axonal routing during mammalian optic chiasm development.Case report.Here, we report a case of congenital myopia, anisometropia, and obesity in a patient with a SLIT2 point mutation. Examination of the patient's skin biopsy revealed abnormalities in elastin and collagen fibrils that suggest an underlying connective tissue disorder. Structural modeling placed the novel mutation (p.D1407G) in the EGF-like domain 8 and was predicted to affect interactions with SLIT2 binding partners.To the authors' knowledge, this is the first report of a SLIT2 variant in the context of these ocular findings.
View details for PubMedID 30111362
- A novel de novo CAPN5 mutation in a patient with inflammatory vitreoretinopathy, hearing loss, and developmental delay COLD SPRING HARBOR MOLECULAR CASE STUDIES 2018; 4 (3)
Extracellular superoxide dismutase 3 (SOD3) regulates oxidative stress at the vitreoretinal interface.
Free radical biology & medicine
Oxidative stress is a pathogenic feature in vitreoretinal disease. However, the ability of the inner retina to manage metabolic waste and oxidative stress is unknown. Proteomic analysis of antioxidants in the human vitreous, the extracellular matrix opposing the inner retina, identified superoxide dismutase-3 (SOD3) that localized to a unique matrix structure in the vitreous base and cortex. To determine the role of SOD3, Sod3-/- mice underwent histological and clinical phenotyping. Although the eyes were structurally normal, at the vitreoretinal interface Sod3-/- mice demonstrated higher levels of 3-nitrotyrosine, a key marker of oxidative stress. Pattern electroretinography also showed physiological signaling abnormalities within the inner retina. Vitreous biopsies and epiretinal membranes collected from patients with diabetic vitreoretinopathy (DVR) and a mouse model of DVR showed significantly higher levels of nitrates and/or 3-nitrotyrosine oxidative stress biomarkers suggestive of SOD3 dysfunction. This study analyzes the molecular pathways that regulate oxidative stress in human vitreous substructures. The absence or dysregulation of the SOD3 antioxidant at the vitreous base and cortex results in increased oxidative stress and tissue damage to the inner retina, which may underlie DVR pathogenesis and other vitreoretinal diseases.
View details for PubMedID 29940351
Fibrin Glue and Internal Limiting Membrane Abrasion for Optic Disc Pit Maculopathy.
Ophthalmic surgery, lasers & imaging retina
2018; 49 (12): e271–e277
To describe a novel surgical technique using pars plana vitrectomy (PPV), internal limiting membrane (ILM) abrasion, and intravitreal fibrin glue for the treatment of optic disc pit maculopathy.Surgical case series technique with scanning electron microscopy (SEM) of human post-mortem eyes.Using SEM, the authors demonstrate the persistent adherence of vitreous fragments to the optic disc following induction of posterior vitreous detachment in human postmortem eyes. The authors describe a surgical technique using PPV, Tano Diamond Dusted Membrane Scraper for an ILM abrasion, intravitreal fibrin glue (Tisseel), and gas-air exchange to seal optic disc pits. The authors report successful long-term visual and anatomical outcomes in three patients.Intravitreal fibrin glue, when combined with ILM abrasion, may be a viable treatment option for optic disc pit maculopathy with good short- and long-term visual acuity outcomes. SEM shows that ILM abrasion removes vitreous fragments, which are persistently adherent and may lead to failure with other interventional techniques. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:e271-e277.].
View details for DOI 10.3928/23258160-20181203-18
View details for PubMedID 30566713
- Therapeutic drug repositioning using personalized proteomics of liquid biopsies JCI INSIGHT 2017; 2 (24)
Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO)
2017; 12 (3)
Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.
View details for DOI 10.1371/journal.pone.0169687
View details for Web of Science ID 000396318300002
View details for PubMedID 28301468
View details for PubMedCentralID PMC5354242
Structural modeling of a novel SLC38A8 mutation that causes foveal hypoplasia.
Molecular Genetics & Genomic Medicine
2017; 5 (3): 202-209
View details for DOI 10.1002/mgg3.266
- Crystal structure of the human calpain-5 catalytic core INT UNION CRYSTALLOGRAPHY. 2017: A31–A32
Structural modeling of a novel SLC38A8 mutation that causes foveal hypoplasia
Molecular Genetics & Genomic Medicine
2017; 5 (3): 202-209
Foveal hypoplasia (FH) in the absence of albinism, aniridia, microphthalmia, or achromatopsia is exceedingly rare, and the molecular basis for the disorder remains unknown. FH is characterized by the absence of both the retinal foveal pit and avascular zone, but with preserved retinal architecture. SLC38A8 encodes a sodium-coupled neutral amino acid transporter with a preference for glutamate as a substrate. SLC38A8 has been linked to FH. Here, we describe a novel mutation to SLC38A8 which causes FH, and report the novel use of OCT-angiography to improve the precision of FH diagnosis. More so, we used computational modeling to explore possible functional effects of known SLC38A8 mutations.Fundus autofluorescence, SD-OCT, and OCT-angiography were used to make the clinical diagnosis. Whole-exome sequencing led to the identification of a novel disease-causing variant in SLC38A8. Computational modeling approaches were used to visualize known SLC38A8 mutations, as well as to predict mutation effects on transporter structure and function.We identified a novel point mutation in SLC38A8 that causes FH. A conclusive diagnosis was made using OCT-angiography, which more clearly revealed retinal vasculature penetrating into the foveal region. Structural modeling of the channel showed the mutation was near previously published mutations, clustered on an extracellular loop. Our modeling also predicted that the mutation destabilizes the protein by altering the electrostatic potential within the channel pore.Our results demonstrate a novel use for OCT-angiography in confirming FH, and also uncover genotype-phenotype correlations of FH-linked SLC38A8 mutations.
View details for DOI 10.1002/mgg3.266
View details for PubMedCentralID PMC5441399
Personalized Proteomics in Proliferative Vitreoretinopathy Implicate Hematopoietic Cell Recruitment and mTOR as a Therapeutic Target
American Journal of Ophthalmology
To profile vitreous cytokine expression of proliferative vitreoretinopathy (PVR) patients DESIGN: Case-control study.Liquid biopsies were collected from two groups: control subjects (n=3) undergoing pars plana vitrectomy to remove an epiretinal membrane (ERM), and test subjects (n=7) with varying degrees of PVR. A high-throughput cytokine screen measured expression of 200 cytokines. Cytokine expression patterns were prospectively validated in separate cohorts of control patients and those with PVR-A, PVR-B, and PVR-C (n=10 for each group). Expression changes were evaluated by ANOVA (significant p-value <0.05), hierarchical cluster algorithm, and pathway analysis, to identify candidate pathways for prospective studies.In PVR vitreous, 29 cytokines were upregulated compared to controls. Early-PVR vitreous showed upregulation of T-cell markers, pro-fibrotic cytokines, and cytokines downstream of mTOR activation (IL-2, IL-6, and IL-13), whereas late PVR vitreous, cytokines driving monocyte responses and stem-cell recruitment (SDF-1) prevailed. Prospective validation confirmed the differential-expression of specific cytokines from PVR-A to C.Early PVR is characterized by activation of T-cells and mTOR signaling, whereas advanced-PVR is characterized by a chronic monocyte response. PVR might be treated by rational repositioning of existing drugs that target mTOR and IL-6. Our analysis demonstrates that successful therapeutic intervention will be highly dependent on the specific therapeutic target and the stage of PVR. This study provides insights into cytokines that will serve as biomarkers and therapeutic targets. These biomarkers will help design clinical trials that intervene at appropriate times.
View details for DOI 10.1016/j.ajo.2017.11.025
Proteomic Analysis of Elevated Intraocular Pressure with Retinal Detachment.
American journal of ophthalmology case reports
2017; 5: 107–10
To report a case of elevated intraocular pressure with retinal detachment.Liquid chromatography and tandem mass spectrometry was performed on the patient aqueous biopsy. Protein levels were analyzed with 1-way analysis of variance (ANOVA) and unbiased clustering. High levels of rod outer segment proteins were not detected, suggesting that this was not a case of Schwartz-Matsuo syndrome. Instead, elevated levels of Hepcidin (HEPC) and Cystatin C (CYTC; candidate biomarkers for primary open angle glaucoma) were detected, suggesting a different, unknown etiology.Molecular diagnoses can differentiate between clinical diagnoses and point to common biomarkers or disease mechanisms.
View details for PubMedID 28825049
View details for PubMedCentralID PMC5560621
Dissection of Human Retina and RPE-Choroid for Proteomic Analysis.
Journal of visualized experiments : JoVE
The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.
View details for PubMedID 29155757
Gene Therapy Restores Mfrp and Corrects Axial Eye Length.
2017; 7 (1): 16151
Hyperopia (farsightedness) is a common and significant cause of visual impairment, and extreme hyperopia (nanophthalmos) is a consequence of loss-of-function MFRP mutations. MFRP deficiency causes abnormal eye growth along the visual axis and significant visual comorbidities, such as angle closure glaucoma, cystic macular edema, and exudative retinal detachment. The Mfrp rd6 /Mfrp rd6 mouse is used as a pre-clinical animal model of retinal degeneration, and we found it was also hyperopic. To test the effect of restoring Mfrp expression, we delivered a wild-type Mfrp to the retinal pigmented epithelium (RPE) of Mfrp rd6 /Mfrp rd6 mice via adeno-associated viral (AAV) gene therapy. Phenotypic rescue was evaluated using non-invasive, human clinical testing, including fundus auto-fluorescence, optical coherence tomography, electroretinography, and ultrasound. These analyses showed gene therapy restored retinal function and normalized axial length. Proteomic analysis of RPE tissue revealed rescue of specific proteins associated with eye growth and normal retinal and RPE function. The favorable response to gene therapy in Mfrp rd6 /Mfrp rd6 mice suggests hyperopia and associated refractive errors may be amenable to AAV gene therapy.
View details for PubMedID 29170418
View details for PubMedCentralID PMC5701072
Small-angle X-ray scattering of calpain-5 reveals a highly open conformation among calpains
JOURNAL OF STRUCTURAL BIOLOGY
2016; 196 (3): 309-318
Calpain-5 is a calcium-activated protease expressed in the retina. Mutations in calpain-5 cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM#193235). The structure of calpain-5 has not been determined, thus hindering the investigation of its proteolytic targets and pathological role in ADNIV. Herein, we report models of the proteolytic core of calpain-5 (mini-calpain-5) containing two globular domains (termed DIIa-IIb) connected by a short, flexible linker, consistent with small-angle X-ray scattering (SAXS) data. Structural modeling in the absence of calcium suggests that mini-calpain-5 adopts a more open conformation when compared to previously determined structures of other calpain cores. This open conformation, achieved by a rotation of DIIa and DIIb with respect to each other, prevents formation of the active site and constrains the enzyme in an inactivated form. The relative domain rotation of 60-100° we found for mini-calpain-5 (a non-classical calpain) is significantly greater than the largest rotation previously observed for a classical calpain (i.e., 55.0° for mini-calpain-9). Together with our prediction that, in the full-length form, a long loop in DIIb (loop C1), a few residues downstream of the inter-domain linker, likely interacts with the shorter, acidic, inactivating loop on domain-III (DIII), these structural insights illuminate the complexity of calpain regulation. Moreover, our studies argue that pursuing higher resolution structural studies are necessary to understand the complex activity regulation prevalent in the calpain family and for the design of specific calpain inhibitors.
View details for DOI 10.1016/j.jsb.2016.07.017
View details for Web of Science ID 000389295000003
View details for PubMedID 27474374
View details for PubMedCentralID PMC5118095
OCULAR HYPERTENSION AFTER INTRAVITREAL DEXAMETHASONE (OZURDEX) SUSTAINED-RELEASE IMPLANT.
Retina (Philadelphia, Pa.)
To evaluate ocular hypertension (OHT) after Ozurdex injection to determine the incidence of OHT, therapy for OHT, and any associative factors such as diagnosis, underlying glaucoma and therapy, or sequential Ozurdex injection(s).Retrospective consecutive case series with patients receiving one or more intravitreal Ozurdex implantations at a tertiary care academic center. Ocular hypertension was defined as a single measurement of ≥30 mmHg or an increase of ≥10 mmHg from baseline.Ninety-four injections in 52 patients (59 eyes) were reviewed. Forty eyes received a single injection, and 19 eyes received multiple injections. Ocular hypertension developed in 14 patients (26.9%). Thirteen patients (25%) had preexisting glaucoma or suspicion of glaucoma, and 6 of these developed OHT. Glaucoma eye drops were initiated after 13 injections (13.8%). Invasive surgery for glaucoma was required in 3 patients (3.2%): all had glaucoma or suspicion of glaucoma (one case was related to neovascular glaucoma and unlikely related to steroid response after Ozurdex). There was no difference in relative intraocular pressure increase (i.e., difference between final follow-up or subsequent intravitreal injection vs. baseline) between single versus multiple Ozurdex injections (P = 0.883).Patients (26.9%) who received Ozurdex developed OHT. Glaucoma or glaucoma-suspicion factors were present in all patients who required invasive surgery for glaucoma. A greater proportion of patients who received multiple injections had an intraocular pressure elevation, but the relative intraocular pressure increase was not significant.
View details for PubMedID 27806001
BESTROPHIN1 mutations cause defective chloride conductance in patient stem cell-derived RPE
HUMAN MOLECULAR GENETICS
2016; 25 (13): 2672-2680
Bestrophin1 (BEST1) is expressed in human retinal pigment epithelium (RPE) and mutations in the BEST1 gene commonly cause retinal dysfunction and macular degeneration. BEST1 is presumed to assemble into a calcium-activated chloride channel and be involved in chloride transport but there is no direct evidence in live human RPE cells to support this idea. To test whether BEST1 functions as a chloride channel in living tissue, BEST1-mutant RPE (R218H, L234P, A243T) were generated from patient-derived induced pluripotent stem cells and compared with wild-type RPE in a retinal environment, using a biosensor that visualizes calcium-induced chloride ion flux in the cell. Calcium stimulation elicited chloride ion export in normal RPE but not in RPE derived from three patients with BEST1 mutations. These data, along with three-dimensional modeling, provide evidence that BEST1 assembles into a key calcium-sensing chloride channel in human RPE.
View details for DOI 10.1093/hmg/ddw126
View details for Web of Science ID 000393064400005
View details for PubMedID 27193166
View details for PubMedCentralID PMC5181636
Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2016; 57 (6): 2509-2521
We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments.CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions.Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains.CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.
View details for DOI 10.1167/iovs.15-18680
View details for Web of Science ID 000378041700018
View details for PubMedID 27152965
View details for PubMedCentralID PMC4868102
Precision Medicine Personalized Proteomics for the Diagnosis and Treatment of Idiopathic Inflammatory Disease
2016; 134 (4): 444-448
To better characterize posterior uveitis, vitreous samples from 15 patients were subjected to antibody arrays, and the expression levels of 200 human cytokines were evaluated. Expression was analyzed by 1-way analysis of variance (significance at P < .01), unsupervised cluster algorithm, and pathway analysis.Unbiased clustering of patients, based on their cytokine expression profile, suggested that particular protein networks and molecular pathways are altered in various forms of uveitis. Expression of interleukin 23 (IL-23), IL-1 receptor I (IL-1RI), IL-17R, tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2), insulinlike growth factor-binding protein 2 (IGFBP-2), nerve growth factor (b-NGF), platelet-derived growth factor receptor β polypeptide (PDGFRb), bone morphogenic protein 4 (BMP-4), and stem cell factor (SCF) constituted a common cytokine signature in the vitreous of patients with uveitis. In 1 patient with progressive, idiopathic visual loss, this last-line analysis implicated retinal autoimmunity, a diagnosis that was validated when her serum sample was found to contain antibodies to S-arrestin, a retinal protein and potent cause of autoimmune retinal degeneration.The analysis identifies a common cytokine signature for posterior uveitis and guides the diagnosis of a patient with idiopathic uveitis. Personalized treatment reversed the visual loss, illustrating how proteomic tools may individualize therapy.
View details for DOI 10.1001/jamaophthalmol.2015.5934
View details for Web of Science ID 000373988900022
View details for PubMedID 26848019
View details for PubMedCentralID PMC4833518
Evidence supporting a critical contribution of intrinsically disordered regions to the biochemical behavior of full-length human HP1 gamma
JOURNAL OF MOLECULAR MODELING
2016; 22 (1)
HP1γ, a non-histone chromatin protein, has elicited significant attention because of its role in gene silencing, elongation, splicing, DNA repair, cell growth, differentiation, and many other cancer-associated processes, including therapy resistance. These characteristics make it an ideal target for developing small drugs for both mechanistic experimentation and potential therapies. While high-resolution structures of the two globular regions of HP1γ, the chromo- and chromoshadow domains, have been solved, little is currently known about the conformational behavior of the full-length protein. Consequently, in the current study, we use threading, homology-based molecular modeling, molecular mechanics calculations, and molecular dynamics simulations to develop models that allow us to infer properties of full-length HP1γ at an atomic resolution level. HP1γ appears as an elongated molecule in which three Intrinsically Disordered Regions (IDRs, 1, 2, and 3) endow this protein with dynamic flexibility, intermolecular recognition properties, and the ability to integrate signals from various intracellular pathways. Our modeling also suggests that the dynamic flexibility imparted to HP1γ by the three IDRs is important for linking nucleosomes with PXVXL motif-containing proteins, in a chromatin environment. The importance of the IDRs in intermolecular recognition is illustrated by the building and study of both IDR2 HP1γ-importin-α and IDR1 and IDR2 HP1γ-DNA complexes. The ability of the three IDRs for integrating cell signals is demonstrated by combined linear motif analyses and molecular dynamics simulations showing that posttranslational modifications can generate a histone mimetic sequence within the IDR2 of HP1γ, which when bound by the chromodomain can lead to an autoinhibited state. Combined, these data underscore the importance of IDRs 1, 2, and 3 in defining the structural and dynamic properties of HP1γ, discoveries that have both mechanistic and potentially biomedical relevance.
View details for DOI 10.1007/s00894-015-2874-z
View details for Web of Science ID 000369312100012
View details for PubMedID 26680990
View details for PubMedCentralID PMC4683166
Functional Characterization of Nupr1L, A Novel p53-Regulated Isoform of the High-Mobility Group (HMG)-Related Protumoral Protein Nupr1
JOURNAL OF CELLULAR PHYSIOLOGY
2015; 230 (12): 2936-2950
We have previously demonstrated a crucial role of nuclear protein 1 (NUPR1) in tumor development and progression. In this work, we report the functional characterization of a novel Nupr1-like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology-based molecular modeling, as well as expression and immunolocalization, studies reveal that NUPR1L displays properties, which are similar to member of the HMG-like family of chromatin regulators, including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53-response elements at positions -37 and -7, respectively. Experiments using reporter assays combined with site-directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well-validated methods to achieve p53 activation. Interestingly, expression of NUPR1L downregulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by downregulating NUPR1 expression. Combined, these experiments constitute the first functional characterization of NUPR1L as a new p53-induced gene, which negatively regulates the protumoral factor NUPR1.
View details for DOI 10.1002/jcp.25022
View details for Web of Science ID 000360378000011
View details for PubMedID 25899918
View details for PubMedCentralID PMC4778741
Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells
2015; 75 (10): 1972-1982
It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.
View details for DOI 10.1158/0008-5472.CAN-14-2761
View details for Web of Science ID 000357332700007
View details for PubMedID 25769722
View details for PubMedCentralID PMC4433621
Evidence supporting the existence of a NUPR1-like family of helix-loop-helix chromatin proteins related to, yet distinct from, AT hook-containing HMG proteins
JOURNAL OF MOLECULAR MODELING
2014; 20 (8)
NUPR1, a small chromatin protein, plays a critical role in cancer development, progression, and resistance to therapy. Here, using a combination of structural bioinformatics and molecular modeling methods, we report several novel findings that enhance our understanding of the biochemical function of this protein. We find that NUPR1 has been conserved throughout evolution, and over time it has undergone duplications and transpositions to form other transcriptional regulators. Using threading, homology-based molecular modeling, molecular mechanics calculations, and molecular dynamics simulations, we generated structural models for four of these proteins: NUPR1a, NUPR1b, NUPR2, and the NUPR-like domain of GTF2-I. Comparative analyses of these models combined with extensive linear motif identification reveal that these four proteins, though similar in their propensities for folding, differ in size, surface changes, and sites amenable for posttranslational modification. Lastly, taking NUPR1a as the paradigm for this family, we built models of a NUPR-DNA complex. Additional structural comparisons revealed that NUPR1 defines a new family of small-groove-binding proteins that share structural features with, yet are distinct from, helix-loop-helix AT-hook-containing HMG proteins. These models and inferences should lead to a better understanding of the function of this group of chromatin proteins, which play a critical role in the development of human malignant diseases.
View details for DOI 10.1007/s00894-014-2357-7
View details for Web of Science ID 000340869400030
View details for PubMedID 25056123
View details for PubMedCentralID PMC4139591
Role for Kruppel-Like Transcription Factor 11 in Mesenchymal Cell Function and Fibrosis
2013; 8 (9)
Krüppel-like factor 11 (KLF11) and the highly homologous KLF10 proteins are transcription factors originating from duplication of the Drosophila melanogaster ancestor cabut. The function of these proteins in epithelial cells has been previously characterized. In the current study, we report a functional role for KLF11 in mesenchymal cells and in mesenchymal cell dysfunction, namely, fibrosis, and subsequently perform a detailed cellular, molecular, and in vivo characterization of this phenomenon. We find that, in cultured mesenchymal cells, enhanced expression of KLF11 results in activated extracellular matrix pathways, including collagen gene silencing and matrix metalloproteinases activation without changes in tissue inhibitors of metalloproteinases. Combined, reporter and chromatin immunoprecipitation assays demonstrate that KLF11 interacts directly with the collagen 1a2 (COL1A2) promoter in mesenchymal cells to repress its activity. Mechanistically, KLF11 regulates collagen gene expression through the heterochromatin protein 1 gene-silencing pathway as mutants defective for coupling to this epigenetic modifier lose the ability to repress COL1A2. Expression studies reveal decreased levels of KLF11 during liver fibrogenesis after chemically induced injury in vivo. Congruently, KLF11(-/-) mice, which should be deficient in the hypothesized anti-fibrogenic brake imposed by this transcription factor, display an enhanced response to liver injury with increased collagen fibril deposition. Thus, KLFs expands the repertoire of transcription factors involved in the regulation of extracellular matrix proteins in mesenchymal cells and define a novel pathway that modulates the fibrogenic response during liver injury.
View details for DOI 10.1371/journal.pone.0075311
View details for Web of Science ID 000324547300078
View details for PubMedID 24069400
View details for PubMedCentralID PMC3775729
Critical role of the HP1-histone methyltransferase pathways in cancer epigenetics
2013; 1 (1): 100-105
View details for DOI 10.1159/000355978