Publications by authors named "Sun Peck"

14 Publications

  • Page 1 of 1

Progression of vertebral bone disease in mucopolysaccharidosis VII dogs from birth to skeletal maturity.

Mol Genet Metab 2021 Aug 15;133(4):378-385. Epub 2021 Jun 15.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, 3450 Hamilton Walk, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, 3450 Hamilton Walk, Philadelphia, PA, USA. Electronic address:

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.
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http://dx.doi.org/10.1016/j.ymgme.2021.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8289741PMC
August 2021

Effects of nanocrystalline hydroxyapatite concentration and skeletal site on bone and cartilage formation in rats.

Acta Biomater 2021 Aug 12;130:485-496. Epub 2021 Jun 12.

Department of Biomedical Engineering, Vanderbilt University, 2201 West End Ave, Nashville TN 37235, United States; Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2201 West End Ave, Nashville TN 37235, United States; Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, 1211 Medical Center Dr., Nashville, TN 37212, United States; Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 1211 Medical Center Dr., Nashville, TN 37217, United States. Electronic address:

Most fractures heal by a combination of endochondral and intramembranous ossification dependent upon strain and vascularity at the fracture site. Many biomaterials-based bone regeneration strategies rely on the use of calcium phosphates such as nano-crystalline hydroxyapatite (nHA) to create bone-like scaffolds. In this study, nHA was dispersed in reactive polymers to form composite scaffolds that were evaluated both in vitro and in vivo. Matrix assays, immunofluorescent staining, and Western blots demonstrated that nHA influenced mineralization and subsequent osteogenesis in a dose-dependent manner in vitro. Furthermore, nHA dispersed in polymeric composites promoted osteogenesis by a similar mechanism as particulated nHA. Scaffolds were implanted into a 2-mm defect in the femoral diaphysis or metaphysis of Sprague-Dawley rats to evaluate new bone formation at 4 and 8 weeks. Two formulations were tested: a poly(thioketal urethane) scaffold without nHA (PTKUR) and a PTKUR scaffold augmented with 22 wt% nHA (22nHA). The scaffolds supported new bone formation in both anatomic sites. In the metaphysis, augmentation of scaffolds with nHA promoted an intramembranous healing response. Within the diaphysis, nHA inhibited endochondral ossification. Immunohistochemistry was performed on cryo-sections of the bone/scaffold interface in which CD146, CD31, Endomucin, CD68, and Myeloperoxidase were evaluated. No significant differences in the infiltrating cell populations were observed. These findings suggest that nHA dispersed in polymeric composites induces osteogenic differentiation of adherent endogenous cells, which has skeletal site-specific effects on fracture healing. STATEMENT OF SIGNIFICANCE: Understanding the mechanism by which synthetic scaffolds promote new bone formation in preclinical models is crucial for bone regeneration applications in the clinic where complex fracture cases are seen. In this study, we found that dispersion of nHA in polymeric scaffolds promoted in vitro osteogenesis in a dose-dependent manner through activation of the PiT1 receptor and subsequent downstream Erk1/2 signaling. While augmentation of polymeric scaffolds with nHA enhanced intramembranous ossification in metaphyseal defects, it inhibited endochondral ossification in diaphyseal defects. Thus, our findings provide new insights into designing synthetic bone grafts that complement the skeletal site-specific fracture healing response.
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http://dx.doi.org/10.1016/j.actbio.2021.05.056DOI Listing
August 2021

Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP).

Dev Biol 2021 02 17;470:136-146. Epub 2020 Nov 17.

Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, 3450 Hamilton Walk, 309A Stemmler Hall, Philadelphia, PA 19104, United States; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, 415 Curie Boulevard, Clinical Research Building, Philadelphia, PA 19104, United States; Center for Research in FOP & Related Disorders, Perelman School of Medicine, University of Pennsylvania, 3450 Hamilton Walk, 309A Stemmler Hall, Philadelphia, PA 19104, United States. Electronic address:

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1 mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1 mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.
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http://dx.doi.org/10.1016/j.ydbio.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035973PMC
February 2021

Molecular profiling of failed endochondral ossification in mucopolysaccharidosis VII.

Bone 2019 11 20;128:115042. Epub 2019 Aug 20.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, 3450 Hamilton Walk, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, 3450 Hamilton Walk, Philadelphia, PA, USA. Electronic address:

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of β-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14 days-of-age (n = 5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14 days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/β-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.
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http://dx.doi.org/10.1016/j.bone.2019.115042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813906PMC
November 2019

Hypoxic Preconditioning Enhances Bone Marrow-Derived Mesenchymal Stem Cell Survival in a Low Oxygen and Nutrient-Limited 3D Microenvironment.

Cartilage 2019 Apr 11:1947603519841675. Epub 2019 Apr 11.

1 Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Objective: Skeletal tissues such as intervertebral disc and articular cartilage possess limited innate potential to regenerate, in part due to their avascularity and low cell density. Despite recent advances in mesenchymal stem cell (MSC)-based disc and cartilage regeneration, key challenges remain, including the sensitivity of these cells to in vivo microenvironmental stress such as low oxygen and limited nutrition. The objective of this study was to investigate whether preconditioning with hypoxia and/or transforming growth factor-β 3 (TGF-β3) can enhance MSC survival and extracellular matrix production in a low oxygen and nutrient-limited microenvironment.

Design: MSCs from multiple bovine donors were preconditioned in monolayer in normoxia or hypoxia, with or without TGF-β3, and the global effects on gene expression were examined using microarrays. Subsequently, the effects of preconditioning on MSC survival and extracellular matrix production were examined using low oxygen and nutrient-limited pellet culture experiments.

Results: Hypoxic preconditioning resulted in upregulation of genes associated with growth, cell-cell signaling, metabolism, and cell stress response pathways, and significantly enhanced MSC survival for all donors in low oxygen and nutrient-limited pellet culture. In contrast, TGF-β3 preconditioning diminished survival. The nature and magnitude of the effects of preconditioning with either hypoxia or TGF-β3 on glycosaminoglycan production were donor dependent.

Conclusions: These results strongly support the use of hypoxic preconditioning to improve postimplantation MSC survival in avascular tissues such as disc and cartilage.
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http://dx.doi.org/10.1177/1947603519841675DOI Listing
April 2019

Neuronal ceroid lipofuscinosis related ER membrane protein CLN8 regulates PP2A activity and ceramide levels.

Biochim Biophys Acta Mol Basis Dis 2019 02 16;1865(2):322-328. Epub 2018 Nov 16.

Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Graduate Biochemistry Group, Kansas State University, Manhattan, Kansas 66506, USA. Electronic address:

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders. CLN8 deficiency causes a subtype of NCL, referred to as CLN8 disease. CLN8 is an ER resident protein with unknown function; however, a role in ceramide metabolism has been suggested. In this report, we identified PP2A and its biological inhibitor I2PP2A as interacting proteins of CLN8. PP2A is one of the major serine/threonine phosphatases in cells and governs a wide range of signaling pathways by dephosphorylating critical signaling molecules. We showed that the phosphorylation levels of several substrates of PP2A, namely Akt, S6 kinase, and GSK3β, were decreased in CLN8 disease patient fibroblasts. This reduction can be reversed by inhibiting PP2A phosphatase activity with cantharidin, suggesting a higher PP2A activity in CLN8-deficient cells. Since ceramides are known to bind and influence the activity of PP2A and I2PP2A, we further examined whether ceramide levels in the CLN8-deficient cells were changed. Interestingly, the ceramide levels were reduced by 60% in CLN8 disease patient cells compared to controls. Furthermore, we observed that the conversion of ER-localized NBD-C6-ceramide to glucosylceramide and sphingomyelin in the Golgi apparatus was not affected in CLN8-deficient cells, indicating transport of ceramides from ER to the Golgi apparatus was normal. A model of how CLN8 along with ceramides affects I2PP2A and PP2A binding and activities is proposed.
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http://dx.doi.org/10.1016/j.bbadis.2018.11.011DOI Listing
February 2019

Quantitative MRI correlates with histological grade in a percutaneous needle injury mouse model of disc degeneration.

J Orthop Res 2018 10 24;36(10):2771-2779. Epub 2018 May 24.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Low back pain due to disc degeneration is a major cause of morbidity and health care expenditures worldwide. While stem cell-based therapies hold promise for disc regeneration, there is an urgent need to develop improved in vivo animal models to further develop and validate these potential treatments. The objectives of this study were to characterize a percutaneous needle injury model of intervertebral disc degeneration in the mouse caudal spine, and compare two non-invasive quantitative imaging techniques, microcomputed tomography and magnetic resonance imaging (MRI), as effective measures of disc degeneration in this model. Percutaneous needle injury of mouse caudal discs was undertaken using different needle sizes and injury types (unilateral or bilateral annulus fibrosus (AF) puncture). Mice were euthanized 4 weeks post-injury, and MRI and microcomputed tomography were used to determine T2 relaxation time of the NP and disc height index, respectively. Disc condition was then further assessed using semi-quantitative histological grading. Bilateral AF puncture with either 27 or 29G needles resulted in significantly lower T2 relaxation times compared to uninjured controls, while disc height index was not significantly affected by any injury type. There was a strong, inverse linear relationship between histological grade and NP T2 relaxation time. In this study, we demonstrated that quantitative MRI can detect disc degeneration in the mouse caudal spine 4 weeks following percutaneous needle injury, and may therefore serve as a surrogate for histology in longitudinal studies of both disc degeneration and cell-based therapies for disc regeneration using this model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2771-2779, 2018.
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http://dx.doi.org/10.1002/jor.24028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200662PMC
October 2018

RNA sequencing identifies gene regulatory networks controlling extracellular matrix synthesis in intervertebral disk tissues.

J Orthop Res 2018 05 10;36(5):1356-1369. Epub 2018 Jan 10.

Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, Minnesota, 55905.

Degenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others.

Clinical Significance: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356-1369, 2018.
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http://dx.doi.org/10.1002/jor.23834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990467PMC
May 2018

Whole Transcriptome Analysis of Notochord-Derived Cells during Embryonic Formation of the Nucleus Pulposus.

Sci Rep 2017 09 5;7(1):10504. Epub 2017 Sep 5.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.
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http://dx.doi.org/10.1038/s41598-017-10692-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585380PMC
September 2017

Pathogenesis and treatment of spine disease in the mucopolysaccharidoses.

Mol Genet Metab 2016 08 4;118(4):232-43. Epub 2016 Jun 4.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, United States; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, United States. Electronic address:

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Skeletal disease is common in MPS patients, with the severity varying both within and between subtypes. Within the spectrum of skeletal disease, spinal manifestations are particularly prevalent. Developmental and degenerative abnormalities affecting the substructures of the spine can result in compression of the spinal cord and associated neural elements. Resulting neurological complications, including pain and paralysis, significantly reduce patient quality of life and life expectancy. Systemic therapies for MPS, such as hematopoietic stem cell transplantation and enzyme replacement therapy, have shown limited efficacy for improving spinal manifestations in patients and animal models. Therefore, there is a pressing need for new therapeutic approaches that specifically target this debilitating aspect of the disease. In this review, we examine how pathological abnormalities affecting the key substructures of the spine - the discs, vertebrae, odontoid process and dura - contribute to the progression of spinal deformity and symptomatic compression of neural elements. Specifically, we review current understanding of the underlying pathophysiology of spine disease in MPS, how the tissues of the spine respond to current clinical and experimental treatments, and discuss future strategies for improving the efficacy of these treatments.
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http://dx.doi.org/10.1016/j.ymgme.2016.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970936PMC
August 2016

Delayed hypertrophic differentiation of epiphyseal chondrocytes contributes to failed secondary ossification in mucopolysaccharidosis VII dogs.

Mol Genet Metab 2015 Nov 26;116(3):195-203. Epub 2015 Sep 26.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient β-glucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal chondrocytes are unable to successfully transition from proliferation to hypertrophy during this critical developmental window. Immunoblotting demonstrated abnormal persistence of Sox9 protein in MPS VII cells between 9 and 14 days-of-age, and biochemical assays revealed abnormally high intra and extracellular GAG content in MPS VII epiphyseal cartilage at as early as 9 days-of-age. In contrast, assessment of vertebral growth plates and primary ossification centers revealed no significant abnormalities at either age. The results of this study establish that failed vertebral bone formation in MPS VII can be traced to the failure of epiphyseal chondrocytes to undergo hypertrophic differentiation at the appropriate developmental stage, and suggest that aberrant processing of Sox9 protein may contribute to this cellular dysfunction. These results also highlight the importance of early diagnosis and therapeutic intervention to prevent the progression of debilitating skeletal disease in MPS patients.
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http://dx.doi.org/10.1016/j.ymgme.2015.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641049PMC
November 2015

The role of N-glycosylation in folding, trafficking, and functionality of lysosomal protein CLN5.

PLoS One 2013 10;8(9):e74299. Epub 2013 Sep 10.

Molecular, Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, United States of America.

CLN5 is a soluble lysosomal protein with unknown function. Mutations in CLN5 lead to neuronal ceroid lipofuscinosis, a group of inherited neurodegenerative disorders that mainly affect children. CLN5 has eight potential N-glycosylation sites based on the Asn-X-Thr/Ser consensus sequence. Through site-directed mutagenesis of individual asparagine residues to glutamine on each of the N-glycosylation consensus sites, we showed that all eight putative N-glycosylation sites are utilized in vivo. Additionally, localization studies showed that the lack of N-glycosylation on certain sites (N179, N252, N304, or N320) caused CLN5 retention in the endoplasmic reticulum, indicating that glycosylation is important for protein folding. Interestingly, one particular mutant, N401Q, is mislocalized to the Golgi, suggesting that N401 is not important for protein folding but essential for CLN5 trafficking to the lysosome. Finally, we analyzed several patient mutations in which N-glycosylation is affected. The N192S patient mutant is localized to the lysosome, indicating that this mutant has a functional defect in the lysosome. Our results suggest that there are functional differences in various N-glycosylation sites of CLN5 which affect folding, trafficking, and lysosomal function of CLN5.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074299PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769244PMC
June 2014

Directed evolution of a small-molecule-triggered intein with improved splicing properties in mammalian cells.

Chem Biol 2011 May;18(5):619-30

Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.

Laboratory-created small-molecule-dependent inteins enable protein structure and function to be controlled posttranslationally in living cells. Previously we evolved inteins that splice efficiently in Saccharomyces cerevisiae only in the presence of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). In mammalian cells, however, these inteins exhibited lower splicing efficiencies and slower splicing in the presence of 4-HT, as well as higher background splicing in the absence of 4-HT. Here we further evolved ligand-dependent inteins in yeast at 30°C and 37°C. The resulting second-generation evolved inteins exhibit substantially improved splicing yields and kinetics. The improvements carried over to mammalian cells, in which the newly evolved inteins spliced with substantially greater (∼2- to 8-fold) efficiency while maintaining low background splicing levels. These second-generation inteins augment the promise of ligand-dependent protein splicing for probing protein function in mammalian cells.
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http://dx.doi.org/10.1016/j.chembiol.2011.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124510PMC
May 2011

A de novo protein binding pair by computational design and directed evolution.

Mol Cell 2011 Apr 31;42(2):250-60. Epub 2011 Mar 31.

Department of Biochemistry, University of Washington, Seattle, WA 98195-7350, USA.

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.
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http://dx.doi.org/10.1016/j.molcel.2011.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102007PMC
April 2011
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