Academic Appointments

  • Instructor, Ophthalmology

Honors & Awards

  • Macular Degeneration Research Postdoctoral Fellow, BrightFocus Foundation (2021-2023)
  • Dean's Postdoctoral Fellowship Finalist, Stanford School of Medicine (Jul. 2021)
  • SUMS Seed Grant Program, Role: Co-PI (PI: Dr. Mahajan), Stanford Dean of Research (Jul. 2019)

Professional Education

  • Postdoctoral Research Fellow, Stanford University, Ophthalmology (2023)
  • Doctor of Philosophy, University of Iowa, Biochemistry (2019)
  • Bachelor of Science, Yonsei University, Systems Biology (2013)

All Publications

  • Identification of highly potent and selective HTRA1 inhibitors. Bioorganic & medicinal chemistry letters Dennis, D. G., Joo Sun, Y., Parsons, D. E., Mahajan, V. B., Smith, M. 2024: 129814


    High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.

    View details for DOI 10.1016/j.bmcl.2024.129814

    View details for PubMedID 38815872

  • Macular dystrophy in Kabuki syndrome due to de novo KMT2D variants: refining the phenotype with multimodal imaging and follow-up over 10 years: insight into pathophysiology. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie Vaclavik, V., Navarro, A., Jacot-Guillarmod, A., Bottani, A., Sun, Y. J., Franco, J. A., Mahajan, V. B., Smirnov, V., Bouvet-Drumare, I. 2024


    BACKGROUND: Kabuki Syndrome is a rare and genetically heterogenous condition with both ophthalmic and systemic complications and typical facial features. We detail the macular phenotype in two unrelated patients with Kabuki syndrome due to de novo nonsense variants in KMT2D, one novel. A follow-up of 10 years is reported. Pathogenicity of both de novo nonsense variants is analyzed.METHODS: Four eyes of two young patients were studied by full clinical examination, kinetic perimetry, short wavelength autofluorescence, full field (ff) ERGs, and spectral-domain optical coherence tomography (SD-OCT). One patient had adaptive optic (AO) imaging. Whole exome sequencing was performed in both patients.RESULTS: Both patients had de novo nonsense variants in KMTD2. One patient had c.14843C>G; p. (Ser4948ter) novel variant and the second c.11119C>T; p. (Arg3707ter). Both had a stable Snellen visual acuity of 0.2-0.3. The retinal multimodal imaging demonstrated abnormalities at the fovea in both eyes: hyperreflectivity to blue light and a well-delimited gap-disruption of ellipsoid and interdigitation layer on OCT. The dark area on AO imaging is presumed to be absent for, or with structural change to photoreceptors. The ff ERGs and kinetic visual fields were normal. The foveal findings remained stable over several years.CONCLUSION: Kabuki syndrome-related maculopathy is a distinct loss of photoreceptors at the fovea as shown by multimodal imaging including, for the first time, AO imaging. This report adds to the literature of only one case with maculopathy with two additional macular dystrophies in patients with Kabuki syndrome. Although underestimated, these cases further raise awareness of the potential impact of retinal manifestations of Kabuki syndrome not only among ophthalmologists but also other healthcare professionals involved in the care of patients with this multisystem disorder.

    View details for DOI 10.1007/s00417-023-06345-1

    View details for PubMedID 38206414

  • Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo. Cell Wolf, J., Rasmussen, D. K., Sun, Y. J., Vu, J. T., Wang, E., Espinosa, C., Bigini, F., Chang, R. T., Montague, A. A., Tang, P. H., Mruthyunjaya, P., Aghaeepour, N., Dufour, A., Bassuk, A. G., Mahajan, V. B. 2023


    Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

    View details for DOI 10.1016/j.cell.2023.09.012

    View details for PubMedID 37863056

  • Unleashing the potential of CRISPR multiplexing: Harnessing Cas12 and Cas13 for precise gene modulation in eye diseases. Vision research Bigini, F., Lee, S. H., Sun, Y. J., Sun, Y., Mahajan, V. B. 2023; 213: 108317


    Gene therapy is a flourishing field with the potential to revolutionize the treatment of genetic diseases. The emergence of CRISPR-Cas9 has significantly advanced targeted and efficient genome editing. Although CRISPR-Cas9 has demonstrated promising potential applications in various genetic disorders, it faces limitations in simultaneously targeting multiple genes. Novel CRISPR systems, such as Cas12 and Cas13, have been developed to overcome these challenges, enabling multiplexing and providing unique advantages. Cas13, in particular, targets mRNA instead of genomic DNA, permitting precise gene expression control and mitigating off-target effects. This review investigates the potential of Cas12 and Cas13 in ocular gene therapy applications, such as suppression of inflammation and cell death. In addition, the capabilities of Cas12 and Cas13 are explored in addressing potential targets related with disease mechanisms such as aberrant isoforms, mitochondrial genes, cis-regulatory sequences, modifier genes, and long non-coding RNAs. Anatomical accessibility and relative immune privilege of the eye provide an ideal organ system for evaluating these novel techniques' efficacy and safety. By targeting multiple genes concurrently, CRISPR-Cas12 and Cas13 systems hold promise for treating a range of ocular disorders, including glaucoma, retinal dystrophies, and age-related macular degeneration. Nonetheless, additional refinement is required to ascertain the safety and efficacy of these approaches in ocular disease treatments. Thus, the development of Cas12 and Cas13 systems marks a significant advancement in gene therapy, offering the potential to devise effective treatments for ocular disorders.

    View details for DOI 10.1016/j.visres.2023.108317

    View details for PubMedID 37722240

  • Conjunctival Swabs Reveal Higher Detection Rate Compared to Schirmer Strips for SARS-CoV-2 RNA Detection in Tears of Hospitalized COVID-19 Patients. Journal of clinical medicine Sabage, L. E., Sun, Y. J., Wolf, J., Sabage, J., Mazzo, A., Santos, C. F., Mahajan, V. B., Manzoni Lourençone, L. F. 2022; 11 (23)


    To determine the prevalence of SARS-CoV-2 in tear samples and to investigate whether it correlates with ocular findings and patients' prognosis in Brazil.Tears were collected using Schirmer strips (SS) and conjunctival swabs (CS) from patients hospitalized with laboratory-confirmed SARS-CoV-2 infection. Samples were analyzed using qRT-PCR. Demographic and clinical data, ocular symptoms, and Schirmer tests (ST) were collected from patients. Charlson Comorbidity Index (CCI) was used to rate comorbidities, and patients were monitored until hospital discharge or death.There were 61 hospitalized patients, 33 of which were diagnosed with COVID-19. Within the confirmed COVID-19 patients, SARS-CoV-2 was detected in 18.2% (n = 6) of CS and 12.1% (n = 4) of SS samples. Subjective and objective parameters for dry eye syndrome (e.g., ST COVID-19: 8.3 ± 6.4mm, non-COVID-19: 8.9 ± 6.6mm, p > 0.05) were comparable between COVID-19 (n = 33) and non-COVID-19 patients (n = 28). Among the 16 COVID-19 patients exhibiting ocular symptoms, only tearing was reported significantly more frequently when tear samples were positive for SARS-CoV-2 (p < 0.05). Strikingly, patients whose tears tested positive for SARS-CoV-2 had significantly inferior CCI (pos.: 34.0 ± 31.8%, neg.: 67.6 ± 36.4%, p < 0.05) and higher mortality rates (pos.: 50.0%, neg.: 7.4%, p < 0.01).SARS-CoV-2 was detected with a prevalence of 18.2% on the ocular surface. Decreased CCI and increased mortality rate in the positive tear group suggests that viral detection may relate to prognosis and highlight the need of personal protective measures for healthcare professionals. Most of the patients, regardless of COVID-19 diagnosis, had low tear production and eye discomfort, possibly pointing to the need for artificial tear use during hospitalization.

    View details for DOI 10.3390/jcm11236929

    View details for PubMedID 36498504

  • Protocol to quantify enzymatic effects on vitreous liquefaction in porcine eyes using a transwell-plate system. STAR protocols Wolf, J., Sabage, L. E., Sun, Y. J., Mahajan, V. B. 2022; 3 (4): 101754


    This protocol describes an exvivo model to quantify enzymatic effects on vitreous liquefaction using porcine eyes in a transwell-plate system via induced syneresis. It provides a standardized dissection process and performs critical steps for gel-liquid separation with high precision, minimal tissue loss, and scalability. The protocol can be applied to other studies investigating vitreous liquefaction or gelatinous tissue analysis and can also serve to study vitreous liquefaction invivo as it may occur during aging or disease progression.

    View details for DOI 10.1016/j.xpro.2022.101754

    View details for PubMedID 36208453

  • Calpains as mechanistic drivers and therapeutic targets for ocular disease. Trends in molecular medicine Vu, J. T., Wang, E., Wu, J., Sun, Y. J., Velez, G., Bassuk, A. G., Lee, S. H., Mahajan, V. B. 2022


    Ophthalmic neurodegenerative diseases encompass a wide array of molecular pathologies unified by calpain dysregulation. Calpains are calcium-dependent proteases that perpetuate cellular death and inflammation when hyperactivated. Calpain inhibition trials in other organs have faced pharmacological challenges, but the eye offers many advantages for the development and testing of targeted molecular therapeutics, including small molecules, peptides, engineered proteins, drug implants, and gene-based therapies. This review highlights structural mechanisms underlying calpain activation, distinct cellular expression patterns, and in vivo models that link calpain hyperactivity to human retinal and developmental disease. Optimizing therapeutic approaches for calpain-mediated eye diseases can help accelerate clinically feasible strategies for treating calpain dysregulation in other diseased tissues.

    View details for DOI 10.1016/j.molmed.2022.05.007

    View details for PubMedID 35641420

  • Expanding the phenotype of TTLL5-associated retinal dystrophy: a case series. Orphanet journal of rare diseases Oh, J. K., Vargas Del Valle, J. G., Lima de Carvalho, J. R., Sun, Y. J., Levi, S. R., Ryu, J., Yang, J., Nagasaki, T., Emanuelli, A., Rasool, N., Allikmets, R., Sparrow, J. R., Izquierdo, N. J., Duncan, J. L., Mahajan, V. B., Tsang, S. H. 2022; 17 (1): 146


    BACKGROUND: Inherited retinal dystrophies describe a heterogeneous group of retinal diseases that lead to the irreversible degeneration of rod and cone photoreceptors and eventual blindness. Recessive loss-of-function mutations in Tubulin Tyrosine Ligase Like 5 (TTLL5) represent a recently described cause of inherited cone-rod and cone dystrophy. This study describes the unusual phenotypes of three patients with autosomal recessive mutations in TTLL5. Examination of these patients included funduscopic evaluation, spectral-domain optical coherence tomography, short-wavelength autofluorescence, and full-field electroretinography (ffERG). Genetic diagnoses were confirmed using whole exome capture. Protein modeling of the identified variants was performed to explore potential genotype-phenotype correlations.RESULTS: Genetic testing revealed five novel variants in TTLL5 in three unrelated patients with retinal dystrophy. Clinical imaging demonstrated features of sectoral cone-rod dystrophy and cone dystrophy, with phenotypic variability seen across all three patients. One patient also developed high-frequency hearing loss during a similar time period as the onset of retinal disease, potentially suggestive of a syndromic disorder. Retinal structure findings were corroborated with functional measures including ffERG findings that supported these diagnoses. Modeling of the five variants suggest that they cause different effects on protein function, providing a potential reason for genotype-phenotype correlation in these patients.CONCLUSIONS: The authors report retinal phenotypic findings in three unrelated patients with novel mutations causing autosomal recessive TTLL5-mediated retinal dystrophy. These findings broaden the understanding of the phenotypes associated with TTLL5-mediated retinal disease and suggest that mutations in TTLL5 should be considered as a potential cause of sectoral retinal dystrophy in addition to cone-rod and cone dystrophies.

    View details for DOI 10.1186/s13023-022-02295-9

    View details for PubMedID 35365235

  • A protocol to inject ocular drug implants into mouse eyes. STAR protocols Lin, C., Sun, Y. J., Lee, S. H., Mujica, E. M., Kunchur, C. R., Wu, M., Yang, J., Jung, Y. S., Chiang, B., Wang, S., Mahajan, V. B. 2022; 3 (1): 101143


    Ocular drug implants (ODIs) are beneficial for treating ocular diseases. However, the lack of a robust injection approach for small-eyed model organisms has been a major technical limitation in developing ODIs. Here, we present a cost-effective, minimally invasive protocol to deliver ODIs into the mouse vitreous called Mouse Implant Intravitreal Injection (MI3). MI3 provides two alternative surgical approaches (air-pressure or plunger) to deliver micro-scaled ODIs into milli-scaled eyes, and expands the preclinical platforms to determine ODIs' efficacy, toxicity, and pharmacokinetics. For complete details on the use and execution of this protocol, please refer to Sun etal. (2021).

    View details for DOI 10.1016/j.xpro.2022.101143

    View details for PubMedID 35141566

  • New COL6A6 Variant Causes Autosomal Dominant Retinitis Pigmentosa in a Four-Generation Family. Investigative ophthalmology & visual science Vaclavik, V., Tiab, L., Sun, Y. J., Mahajan, V. B., Moulin, A., Allaman-Pillet, N., Munier, F. L., Schorderet, D. F. 2022; 63 (3): 23


    Purpose: To report that variants in the gene for a large lamina basal component protein, COL6A6 (collagen type VI alpha 6 chain, Col6alpha6), linked to chromosome 3p22.1 causes retinitis pigmentosa (RP) in patients with autosomal dominant transmission (adRP).Methods: A positional-cloning approach, whole exome sequencing, and modeling were used. The proband and several affected family members have been phenotyped and followed for over 12 years.Results: A heterozygous missense variant, c.509C>G (p. Ser170Cys) in exon 2 of COL6A6 (comprised of 36 exons and 2236 amino acids), was observed in a four- generation family and is likely to cause the adRP phenotype. It was identified in 10 affected members. All affected family members had a distinct phenotype: late-onset rod cone dystrophy, with good retained visual acuity, until their late 70s. Immunohistochemistry of human retina showed a dot-like signal at the base of the inner segments of photoreceptors and outer plexiform layer (OPL). The structural modeling of the N7 domain of Col6alpha6 suggests that the mutant might result in the abnormal cellular localization of collagen VI or malformation of collagen fibers resulting in the loss of its unique filament structure.Conclusions: COL6A6 is widely expressed in human tissues and evolutionary conserved. It is thought to interact with a range of extracellular matrix components. Our findings suggest that this form of RP has long-term useful central visual acuity and a mild progression, which are important considerations for patient counseling.

    View details for DOI 10.1167/iovs.63.3.23

    View details for PubMedID 35333290

  • An intravitreal implant injection method for sustained drug delivery into mouse eyes. Cell reports methods Sun, Y. J., Lin, C., Wu, M., Lee, S. H., Yang, J., Kunchur, C. R., Mujica, E. M., Chiang, B., Jung, Y. S., Wang, S., Mahajan, V. B. 2021; 1 (8)


    Using small molecule drugs to treat eye diseases carries benefits of specificity, scalability, and transportability, but their efficacy is significantly limited by a fast intraocular clearance rate. Ocular drug implants (ODIs) present a compelling means for the slow and sustained release of small molecule drugs inside the eye. However, methods are needed to inject small molecule ODIs into animals with small eyes, such as mice, which are the primary genetic models for most human ocular diseases. Consequently, it has not been possible to fully investigate efficacy and ocular pharmacokinetics of ODIs. Here, we present a robust, cost-effective, and minimally invasive method called "mouse implant intravitreal injection" (MI3) to deliver ODIs into mouse eyes. This method will expand ODI research to cover the breadth of human eye diseases modeled in mice.

    View details for DOI 10.1016/j.crmeth.2021.100125

    View details for PubMedID 35128514

  • A Fluorescence-Based Assay to Determine PDZ-Ligand Binding Thermodynamics. Methods in molecular biology (Clifton, N.J.) Sun, Y. J., Fuentes, E. J. 2021; 2256: 137-148


    Postsynaptic density-95, disks-large, and zonula occludens-1 (PDZ) domain interactions with cognate linear binding motifs (i.e., PDZ-binding motifs or PBMs) are important for many biological processes and can be pathological when disrupted. There are hundreds of PDZ-PBM interactions reported but few have been quantitatively determined. Moreover, PDZ-PBM interactions have been identified as potential therapeutic targets. To thoroughly understand PDZ-PBM binding energetics and their specificity, we have developed a sensitive and quantitative equilibrium binding assay. Here, we describe a protocol for determining PDZ-PBM binding energetics using fluorescence anisotropy-based methodology.

    View details for DOI 10.1007/978-1-0716-1166-1_8

    View details for PubMedID 34014520

  • Peptidomimetics Therapeutics for Retinal Disease. Biomolecules Parsons, D. E., Lee, S. H., Sun, Y. J., Velez, G., Bassuk, A. G., Smith, M., Mahajan, V. B. 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

  • Structure-based phylogeny identifies Avoralstat as a TMPRSS2 inhibitor that prevents SARS-CoV-2 infection in mice. The Journal of clinical investigation Sun, Y. J., Velez, G. n., Parsons, D. E., Li, K. n., Ortiz, M. E., Sharma, S. n., McCray, P. B., Bassuk, A. G., Mahajan, V. B. 2021


    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

  • A physics-based energy function allows the computational redesign of a PDZ domain. Scientific reports Opuu, V., Sun, Y. J., Hou, T., Panel, N., Fuentes, E. J., Simonson, T. 2020; 10 (1): 11150


    Computational protein design (CPD) can address the inverse folding problem, exploring a large space of sequences and selecting ones predicted to fold. CPD was used previously to redesign several proteins, employing a knowledge-based energy function for both the folded and unfolded states. We show that a PDZ domain can be entirely redesigned using a "physics-based" energy for the folded state and a knowledge-based energy for the unfolded state. Thousands of sequences were generated by Monte Carlo simulation. Three were chosen for experimental testing, based on their low energies and several empirical criteria. All three could be overexpressed and had native-like circular dichroism spectra and 1D-NMR spectra typical of folded structures. Two had upshifted thermal denaturation curves when a peptide ligand was present, indicating binding and suggesting folding to a correct, PDZ structure. Evidently, the physical principles that govern folded proteins, with a dash of empirical post-filtering, can allow successful whole-protein redesign.

    View details for DOI 10.1038/s41598-020-67972-w

    View details for PubMedID 32636412

    View details for PubMedCentralID PMC7341745

  • Novel PRPF31 gene loss of function for retinitis pigmentosa 11 Chemudupati, T., Sun, Y., Yang, J., Ng, B., Mahajan, V. B. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020
  • Structural Insights into the Unique Activation Mechanisms of a Non-classical Calpain and Its Disease-Causing Variants. Cell reports Velez, G., Sun, Y. J., Khan, S., Yang, J., Herrmann, J., Chemudupati, T., MacLaren, R. E., Gakhar, L., Wakatsuki, S., Bassuk, A. G., Mahajan, V. B. 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

  • SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility JOURNAL OF CELL BIOLOGY Awadia, S., Hug, F., Arnold, T. H., Goicoechea, S. M., Sun, Y., Hou, T., Kreider-Letterman, G., Massimi, P., Banks, L., Fuentes, E. J., Miller, A. L., Garcia-Mata, R. 2019; 218 (8): 2699–2725


    The canonical Scribble polarity complex is implicated in regulation of epithelial junctions and apical polarity. Here, we show that SGEF, a RhoG-specific GEF, forms a ternary complex with Scribble and Dlg1, two members of the Scribble complex. SGEF targets to apical junctions in a Scribble-dependent fashion and functions in the regulation of actomyosin-based contractility and barrier function at tight junctions as well as E-cadherin-mediated formation of adherens junctions. Surprisingly, SGEF does not control the establishment of polarity. However, in 3D cysts, SGEF regulates the formation of a single open lumen. Interestingly, SGEF's nucleotide exchange activity regulates the formation and maintenance of adherens junctions, and in cysts the number of lumens formed, whereas SGEF's scaffolding activity is critical for regulation of actomyosin contractility and lumen opening. We propose that SGEF plays a key role in coordinating junctional assembly and actomyosin contractility by bringing together Scribble and Dlg1 and targeting RhoG activation to cell-cell junctions.

    View details for DOI 10.1083/jcb.201811114

    View details for Web of Science ID 000478788200019

    View details for PubMedID 31248911

    View details for PubMedCentralID PMC6683736

  • Biochemical and Structural Characterization of De Novo Designed PDZ Domains Fuentes, E. J., Sun, Y., Sternke, M., Opuu, V., Panel, N., Barrick, D., Simonson, T. CELL PRESS. 2019: 320A
  • Novel mutations in the 3-box motif of the BACK domain of KLHL7 associated with nonsyndromic autosomal dominant retinitis pigmentosa. Orphanet journal of rare diseases Oh, J. K., Lima de Carvalho, J. R., Sun, Y. J., Ragi, S. n., Yang, J. n., Levi, S. R., Ryu, J. n., Bassuk, A. G., Mahajan, V. B., Tsang, S. H. 2019; 14 (1): 295


    Mutations in the Kelch-like protein 7 (KLHL7) represent a recently described and, to date, poorly characterized etiology of inherited retinal dystrophy. Dominant mutations in KLHL7 are a cause of isolated, non-syndromic retinitis pigmentosa (RP). In contrast, recessive loss-of-function mutations are known to cause Crisponi or Bohring-Opitz like cold induced sweating syndrome-3 (BOS-3). In this study, the phenotype and progression of five unrelated patients with KLHL7 mediated autosomal dominant RP (adRP) are characterized. Clinical evaluation of these patients involved a complete ophthalmic exam, full-field electroretinography (ffERG), and imaging, including fundus photography, spectral domain optical coherence tomography (SD-OCT), short wavelength fundus autofluorescence (SW-AF), and near-infrared fundus autofluorescence (NIR-AF). Molecular diagnoses were performed using whole-exome sequencing or gene panel testing. Disease progression was monitored in three patients with available data for a mean follow up time of 4.5 ± 2.9 years. Protein modeling was performed for all variants found in this study in addition to those documented in the literature for recessive loss-of-function alleles causing Crisponi or Bohring-Opitz like cold-induced sweating syndrome.Genetic testing in three patients identified two novel variants within the 3-box motif of the BACK domain: c.472 T > C:p.(Cys158Arg) and c.433A > T:p.(Asn145Tyr). Clinical imaging demonstrated hyperautofluorescent ring formation on both SW-AF and NIR-AF in three patients, with diffuse peripheral and peripapillary atrophy seen in all but one case. SD-OCT demonstrated a phenotypic spectrum, from parafoveal atrophy of the outer retina with foveal sparing to widespread retinal thinning and loss of photoreceptors. Incidence of cystoid macular edema was high with four of five patients affected. Protein modeling of dominant alleles versus recessive loss-of-function alleles showed dominant alleles localized to the BTB and BACK domains while recessive alleles were found in the Kelch domain.We report the phenotype in five patients with KLHL7 mediated adRP, two novel coding variants, and imaging biomarkers using SW-AF and NIR-AF. These findings may influence future gene-based therapies for adRP and pave the way for mechanistic studies that elucidate the pathogenesis of KLHL7-mediated RP.

    View details for DOI 10.1186/s13023-019-1275-2

    View details for PubMedID 31856884

  • A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain. Frontiers in molecular biosciences Panel, N., Sun, Y. J., Fuentes, E. J., Simonson, T. 2017; 4: 65


    PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or "PB/LIE" free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

    View details for DOI 10.3389/fmolb.2017.00065

    View details for PubMedID 29018806

    View details for PubMedCentralID PMC5623046

  • Distinct Roles for Conformational Dynamics in Protein-Ligand Interactions. Structure (London, England : 1993) Liu, X., Speckhard, D. C., Shepherd, T. R., Sun, Y. J., Hengel, S. R., Yu, L., Fowler, C. A., Gakhar, L., Fuentes, E. J. 2016; 24 (12): 2053-2066


    Conformational dynamics has an established role in enzyme catalysis, but its contribution to ligand binding and specificity is largely unexplored. Here we used the Tiam1 PDZ domain and an engineered variant (QM PDZ) with broadened specificity to investigate the role of structure and conformational dynamics in molecular recognition. Crystal structures of the QM PDZ domain both free and bound to ligands showed structural features central to binding (enthalpy), while nuclear-magnetic-resonance-based methyl relaxation experiments and isothermal titration calorimetry revealed that conformational entropy contributes to affinity. In addition to motions relevant to thermodynamics, slower microsecond to millisecond switching was prevalent in the QM PDZ ligand-binding site consistent with a role in ligand specificity. Our data indicate that conformational dynamics plays distinct and fundamental roles in tuning the affinity (conformational entropy) and specificity (excited-state conformations) of molecular interactions. More broadly, our results have important implications for the evolution, regulation, and design of protein-ligand interactions.

    View details for DOI 10.1016/j.str.2016.08.019

    View details for PubMedID 27998539

    View details for PubMedCentralID PMC5488749