Publications by authors named "Craig W Vander Kooi"

62 Publications

An empirical pipeline for personalized diagnosis of Lafora disease mutations.

iScience 2021 Nov 13;24(11):103276. Epub 2021 Oct 13.

Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.

Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either encoding the glycogen phosphatase laforin, or , encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline enabled rapid characterization and classification of newly identified mutations, providing clinicians and researchers genetic information to guide treatment of LD patients.
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http://dx.doi.org/10.1016/j.isci.2021.103276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8564118PMC
November 2021

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Biochemistry 2021 08 28;60(31):2425-2435. Epub 2021 Jul 28.

Department of Chemistry, Skidmore College, Saratoga Springs, New York 12866, United States.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.
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http://dx.doi.org/10.1021/acs.biochem.1c00307DOI Listing
August 2021

Brain glycogen serves as a critical glucosamine cache required for protein glycosylation.

Cell Metab 2021 Jul 26;33(7):1404-1417.e9. Epub 2021 May 26.

Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.

Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.
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http://dx.doi.org/10.1016/j.cmet.2021.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266748PMC
July 2021

The 6th International Lafora Epilepsy Workshop: Advances in the search for a cure.

Epilepsy Behav 2021 06 1;119:107975. Epub 2021 May 1.

Department of Molecular and Cellular Biochemistry, Epilepsy and Brain Metabolism Alliance, and Epilepsy Research Center, University of Kentucky College of Medicine, Lexington, KY 40536, USA; Lafora Epilepsy Cure Initiative (LECI), USA. Electronic address:

Lafora disease (LD) is a fatal childhood dementia with severe epilepsy and also a glycogen storage disease that is caused by recessive mutations in either the EPM2A or EPM2B genes. Aberrant, cytoplasmic carbohydrate aggregates called Lafora bodies (LBs) are both a hallmark and driver of the disease. The 6 International Lafora Epilepsy Workshop was held online due to the pandemic. Nearly 300 clinicians, academic and industry scientists, trainees, NIH representatives, and LD friends and family members participated in the event. Speakers covered aspects of LD including progress towards the clinic, the importance of establishing clinical progression, translational progress with repurposed drugs and additional pre-clinical therapies, and novel discoveries that define foundational LD mechanisms.
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http://dx.doi.org/10.1016/j.yebeh.2021.107975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154720PMC
June 2021

Generation and characterization of a laforin nanobody inhibitor.

Clin Biochem 2021 Jul 5;93:80-89. Epub 2021 Apr 5.

Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, United States; Lafora Epilepsy Cure Initiative, University of Kentucky College of Medicine, Lexington, KY 40536, United States. Electronic address:

Objectives: Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor.

Design And Methods: We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin's catalytic activity.

Results: Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin's phosphatase activity.

Conclusions: The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.
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http://dx.doi.org/10.1016/j.clinbiochem.2021.03.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217207PMC
July 2021

Multimodal imaging and genetic findings in a case of ARSG-related atypical Usher syndrome.

Ophthalmic Genet 2021 06 25;42(3):338-343. Epub 2021 Feb 25.

Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, Kentucky, USA.

: Atypical Usher syndrome has recently been associated with arylsulfatase G ( variants. In these cases, characteristic findings include progressive sensorineural hearing loss (SNHL) without vestibular involvement and ring-shaped late-onset retinitis pigmentosa (RP). One patient with atypical Usher syndrome and a novel homozygous variant was included in this study. The patient underwent a comprehensive ophthalmic examination, including multimodal imaging and genetic testing. A 60-year-old male of Persian decent presented to our clinic with a history of 20 years of progressive SNHL, and 10 years of progressive peripheral vision loss and pigmentary retinopathy. Consistent with previous reports of -related atypical Usher syndrome, fundus examination revealed ring-shaped retinal hyperpigmentation and fundus autofluorescence (FAF) demonstrated a six-zone pattern of autofluorescence. Optical coherence tomography (OCT) showed extensive cystoid spaces concentrated in the ganglion cell layer. Widefield OCT angiography at the level of the choriocapillaris showed signs of atrophy that corresponded to the FAF hypofluorescent zone. The patient was homozygous for a novel variant c. 1270 C > T, p. Arg424Cys. We report a novel variant in a case of atypical Usher syndrome and describe multimodal imaging findings that further characterize the effect of in the pathogenesis of atypical Usher syndrome.
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http://dx.doi.org/10.1080/13816810.2021.1891552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154670PMC
June 2021

Structural dissimilarity from self drives neoepitope escape from immune tolerance.

Nat Chem Biol 2020 11 17;16(11):1269-1276. Epub 2020 Aug 17.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.

T-cell recognition of peptides incorporating nonsynonymous mutations, or neoepitopes, is a cornerstone of tumor immunity and forms the basis of new immunotherapy approaches including personalized cancer vaccines. Yet as they are derived from self-peptides, the means through which immunogenic neoepitopes overcome immune self-tolerance are often unclear. Here we show that a point mutation in a non-major histocompatibility complex anchor position induces structural and dynamic changes in an immunologically active ovarian cancer neoepitope. The changes pre-organize the peptide into a conformation optimal for recognition by a neoepitope-specific T-cell receptor, allowing the receptor to bind the neoepitope with high affinity and deliver potent T-cell signals. Our results emphasize the importance of structural and physical changes relative to self in neoepitope immunogenicity. Considered broadly, these findings can help explain some of the difficulties in identifying immunogenic neoepitopes from sequence alone and provide guidance for developing novel, neoepitope-based personalized therapies.
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http://dx.doi.org/10.1038/s41589-020-0610-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210748PMC
November 2020

Design, synthesis, and evaluation of a novel benzamidine-based inhibitor of VEGF-C binding to Neuropilin-2.

Bioorg Chem 2020 07 16;100:103856. Epub 2020 Apr 16.

Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, United States. Electronic address:

The Neuropilin (Nrp) family of cell surface receptors have key physiological and pathological functions. Nrp2 is of particular interest due to its involvement in tumor metastasis. Currently, peptide and small molecule inhibitors that target Nrp utilize arginine-based molecules which have limitations due to high inherent flexibility and issues related to stability. Further, there are no known small molecule inhibitors specific for Nrp2. Recent molecular insights identify a key ligand binding region in the b1 domain of Nrp2 responsible for binding the C-terminus of its cognate ligand VEGF-C. Based on this, we report the discovery of a novel benzamidine-based inhibitor that functions through competitive inhibition of VEGF-C binding to Nrp2. Further, we have explored inhibitor functionality and selectivity by defining its structure-activity relationship (SAR) providing valuable insights on this benzamidine-based family of Nrp2 inhibitors. This study provides the basis for further development of a potent and specific small molecule inhibitor that competitively targets pathological Nrp2 function.
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http://dx.doi.org/10.1016/j.bioorg.2020.103856DOI Listing
July 2020

N-glycosylation-defective splice variants of neuropilin-1 promote metastasis by activating endosomal signals.

Nat Commun 2019 08 16;10(1):3708. Epub 2019 Aug 16.

Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, 40506, USA.

Neuropilin-1 (NRP1) is an essential transmembrane receptor with a variety of cellular functions. Here, we identify two human NRP1 splice variants resulting from the skipping of exon 4 and 5, respectively, in colorectal cancer (CRC). Both NRP1 variants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes. This increased endocytic trafficking of the two NRP1 variants, upon HGF stimulation, is due to loss of N-glycosylation at the Asn150 or Asn261 site, respectively. Moreover, these NRP1 variants enhance interactions with the Met and β1-integrin receptors, resulting in Met/β1-integrin co-internalization and co-accumulation on endosomes. This provides persistent signals to activate the FAK/p130Cas pathway, thereby promoting CRC cell migration, invasion and metastasis. Blocking endocytosis or endosomal Met/β1-integrin/FAK signaling profoundly inhibits the oncogenic effects of both NRP1 variants. These findings reveal an important role for these NRP1 splice variants in the regulation of endocytic trafficking for cancer cell dissemination.
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http://dx.doi.org/10.1038/s41467-019-11580-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697747PMC
August 2019

Central Nervous System Delivery and Biodistribution Analysis of an Antibody-Enzyme Fusion for the Treatment of Lafora Disease.

Mol Pharm 2019 09 2;16(9):3791-3801. Epub 2019 Aug 2.

Valerion Therapeutics , Concord , Massachusetts 01742 , United States.

Lafora disease (LD) is a fatal juvenile epilepsy characterized by the accumulation of aberrant glucan aggregates called Lafora bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay has been developed to detect VAL-0417 post-treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found that intracerebroventricular administration provided robust CNS delivery when compared to intrathecal administration. These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189208PMC
September 2019

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies.

J Vis Exp 2019 01 26(143). Epub 2019 Jan 26.

Department of Molecular and Cellular Biochemistry, University of Kentucky;

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.
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http://dx.doi.org/10.3791/58471DOI Listing
January 2019

Inhibition of Semaphorin3A Promotes Ocular Dominance Plasticity in the Adult Rat Visual Cortex.

Mol Neurobiol 2019 Sep 31;56(9):5987-5997. Epub 2019 Jan 31.

Institute of Neuroscience, National Research Council CNR, Via Moruzzi, 1, 56124, Pisa, Italy.

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity.
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http://dx.doi.org/10.1007/s12035-019-1499-0DOI Listing
September 2019

Improving T Cell Receptor On-Target Specificity via Structure-Guided Design.

Mol Ther 2019 02 14;27(2):300-313. Epub 2018 Dec 14.

Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, USA. Electronic address:

T cell receptors (TCRs) have emerged as a new class of immunological therapeutics. However, though antigen specificity is a hallmark of adaptive immunity, TCRs themselves do not possess the high specificity of monoclonal antibodies. Although a necessary function of T cell biology, the resulting cross-reactivity presents a significant challenge for TCR-based therapeutic development, as it creates the potential for off-target recognition and immune toxicity. Efforts to enhance TCR specificity by mimicking the antibody maturation process and enhancing affinity can inadvertently exacerbate TCR cross-reactivity. Here we demonstrate this concern by showing that even peptide-targeted mutations in the TCR can introduce new reactivities against peptides that bear similarity to the original target. To counteract this, we explored a novel structure-guided approach for enhancing TCR specificity independent of affinity. Tested with the MART-1-specific TCR DMF5, our approach had a small but discernible impact on cross-reactivity toward MART-1 homologs yet was able to eliminate DMF5 cross-recognition of more divergent, unrelated epitopes. Our study provides a proof of principle for the use of advanced structure-guided design techniques for improving TCR specificity, and it suggests new ways forward for enhancing TCRs for therapeutic use.
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http://dx.doi.org/10.1016/j.ymthe.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369632PMC
February 2019

Oligomerization and carbohydrate binding of glucan phosphatases.

Anal Biochem 2018 12 3;563:51-55. Epub 2018 Oct 3.

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, 40536, USA; Center for Structural Biology, University of Kentucky, Lexington, KY, 40536, USA. Electronic address:

Glucan phosphatases are a unique subset of the phosphatase family that bind to and dephosphorylate carbohydrate substrates. Family members are found in diverse organisms ranging from single-cell red algae to humans. The nature of their functional oligomerization has been a source of considerable debate. We demonstrate that the human laforin protein behaves aberrantly when subjected to Size Exclusion Chromotography (SEC) analysis due to interaction with the carbohydrate-based matrix. This interaction complicates the analysis of laforin human disease mutations. Herein, we show that SEC with Multi-Angle static Light Scattering (SEC-MALS) provides a method to robustly define the oligomerization state of laforin and laforin variants. We further analyzed glucan phosphatases from photosynthetic organisms to define if this interaction was characteristic of all glucan phosphatases. Starch EXcess-four (SEX4) from green plants was found to lack significant interaction with the matrix and instead exists as a monomer. Conversely, Cm-laforin, from red algae, exists as a monomer in solution while still exhibiting significant interaction with the matrix. These data demonstrate a range of oligomerization behaviors of members of the glucan phosphatase family, and establish SEC-MALS as a robust methodology to quantify and compare oligomerization states between different proteins and protein variants in this family.
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http://dx.doi.org/10.1016/j.ab.2018.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233729PMC
December 2018

T cell receptor cross-reactivity expanded by dramatic peptide-MHC adaptability.

Nat Chem Biol 2018 10 17;14(10):934-942. Epub 2018 Sep 17.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.

T cell receptor cross-reactivity allows a fixed T cell repertoire to respond to a much larger universe of potential antigens. Recent work has emphasized the importance of peptide structural and chemical homology, as opposed to sequence similarity, in T cell receptor cross-reactivity. Surprisingly, though, T cell receptors can also cross-react between ligands with little physiochemical commonalities. Studying the clinically relevant receptor DMF5, we demonstrate that cross-recognition of such divergent antigens can occur through mechanisms that involve heretofore unanticipated rearrangements in the peptide and presenting MHC protein, including binding-induced peptide register shifts and extensions from MHC peptide binding grooves. Moreover, cross-reactivity can proceed even when such dramatic rearrangements do not translate into structural or chemical molecular mimicry. Beyond demonstrating new principles of T cell receptor cross-reactivity, our results have implications for efforts to predict and control T cell specificity and cross-reactivity and highlight challenges associated with predicting T cell reactivities.
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http://dx.doi.org/10.1038/s41589-018-0130-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371774PMC
October 2018

How an alloreactive T-cell receptor achieves peptide and MHC specificity.

Proc Natl Acad Sci U S A 2017 06 1;114(24):E4792-E4801. Epub 2017 Jun 1.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556;

T-cell receptor (TCR) allorecognition is often presumed to be relatively nonspecific, attributable to either a TCR focus on exposed major histocompatibility complex (MHC) polymorphisms or the degenerate recognition of allopeptides. However, paradoxically, alloreactivity can proceed with high peptide and MHC specificity. Although the underlying mechanisms remain unclear, the existence of highly specific alloreactive TCRs has led to their use as immunotherapeutics that can circumvent central tolerance and limit graft-versus-host disease. Here, we show how an alloreactive TCR achieves peptide and MHC specificity. The HCV1406 TCR was cloned from T cells that expanded when a hepatitis C virus (HCV)-infected HLA-A2 individual received an HLA-A2 liver allograft. HCV1406 was subsequently shown to recognize the HCV nonstructural protein 3 (NS3):1406-1415 epitope with high specificity when presented by HLA-A2. We show that NS3/HLA-A2 recognition by the HCV1406 TCR is critically dependent on features unique to both the allo-MHC and the NS3 epitope. We also find cooperativity between structural mimicry and a crucial peptide "hot spot" and demonstrate its role, along with the MHC, in directing the specificity of allorecognition. Our results help explain the paradox of specificity in alloreactive TCRs and have implications for their use in immunotherapy and related efforts to manipulate TCR recognition, as well as alloreactivity in general.
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http://dx.doi.org/10.1073/pnas.1700459114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474831PMC
June 2017

Plate-Based Assay for Measuring Direct Semaphorin-Neuropilin Interactions.

Methods Mol Biol 2017 ;1493:73-87

Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, 741 South Limestone Avenue, BBSRB B263, Lexington, KY, 40536, USA.

The semaphorins are an essential family of axon guidance molecules that can be either secreted or are transmembrane proteins. Class 3 semaphorin (Sema3) family members are secreted and provide long-range guidance cues through two receptor families: neuropilins (Nrp) and plexins. Nrp is uniquely required for high-affinity Sema3 binding and signaling. Therefore, characterizing the molecular details of the Sema3/Nrp interaction is important for understanding the broader physiological and pathological role of the Sema3 family of proteins. Here we describe an in vitro plate-based binding assay for characterization of the Sema3/Nrp interaction. This assay utilizes Nrp-affinity plates and an alkaline phosphatase (AP)-Sema3 fusion to rapidly measure direct Sema3/Nrp binding. This assay can be used to measure receptor-ligand binding, the contribution of different domains, and exogenous factors, and to characterize competitive ligand binding.
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http://dx.doi.org/10.1007/978-1-4939-6448-2_5DOI Listing
February 2018

Structural biology of glucan phosphatases from humans to plants.

Curr Opin Struct Biol 2016 10 4;40:62-69. Epub 2016 Aug 4.

Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, KY 40536, United States. Electronic address:

Glucan phosphatases are functionally conserved at the enzymatic level, dephosphorylating glycogen in animals and starch in plants. The human glucan phosphatase laforin is the founding member of the family and it is comprised of a carbohydrate binding module (CBM) domain followed by a dual specificity phosphatase (DSP) domain. Plants encode two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a DSP domain followed by a CBM domain, while LSF2 contains a DSP domain and lacks a CBM. This review demonstrates how glucan phosphatase function is conserved and highlights how each family member employs a unique mechanism to bind and dephosphorylate glucan substrates.
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http://dx.doi.org/10.1016/j.sbi.2016.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161650PMC
October 2016

Structural mechanisms of plant glucan phosphatases in starch metabolism.

FEBS J 2016 07 28;283(13):2427-47. Epub 2016 Mar 28.

Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, KY, USA.

Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two glucan phosphatases, called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases, and outlines how they are uniquely adapted to perform their cellular functions. We outline the physical mechanisms used by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan-binding platform comprising its dual-specificity phosphatase domain and carbohydrate-binding module, while LSF2 utilizes surface binding sites. SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic dual-specificity phosphatase domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2, and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism and protein-glucan interaction, thereby providing a framework for their application in both agricultural and industrial settings.
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http://dx.doi.org/10.1111/febs.13703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935604PMC
July 2016

P-Rex1 Promotes Resistance to VEGF/VEGFR-Targeted Therapy in Prostate Cancer.

Cell Rep 2016 Mar 25;14(9):2193-2208. Epub 2016 Feb 25.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN(pc-/-) transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity of prostate tumors to bevacizumab. These data reveal that prostate tumors harbor cells with stem cell properties that are resistant to inhibitors of VEGF/VEGFR signaling. Combining the use of available VEGF/VEGFR-targeted therapies with P-Rex1 or Rac1 inhibition should improve the efficacy of these therapies significantly.
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http://dx.doi.org/10.1016/j.celrep.2016.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791963PMC
March 2016

Structure and functions of angiotensinogen.

Hypertens Res 2016 Jul 18;39(7):492-500. Epub 2016 Feb 18.

Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY, USA.

Angiotensinogen (AGT) is the sole precursor of all angiotensin peptides. Although AGT is generally considered as a passive substrate of the renin-angiotensin system, there is accumulating evidence that the regulation and functions of AGT are intricate. Understanding the diversity of AGT properties has been enhanced by protein structural analysis and animal studies. In addition to whole-body genetic deletion, AGT can be regulated in vivo by cell-specific procedures, adeno-associated viral approaches and antisense oligonucleotides. Indeed, the availability of these multiple manipulations of AGT in vivo has provided new insights into the multifaceted roles of AGT. In this review, the combination of structural and functional studies is highlighted to focus on the increasing recognition that AGT exerts effects beyond being a sole provider of angiotensin peptides.
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http://dx.doi.org/10.1038/hr.2016.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935807PMC
July 2016

The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis.

Cell Rep 2016 Jan 14;14(3):534-546. Epub 2016 Jan 14.

Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37240, USA. Electronic address:

F-BAR proteins link cellular membranes to the actin cytoskeleton in many biological processes. Here we investigated the function of the Schizosaccharomyces pombe Imp2 F-BAR domain in cytokinesis and find that it is critical for Imp2's role in contractile ring constriction and disassembly. To understand mechanistically how the F-BAR domain functions, we determined its structure, elucidated how it interacts with membranes, and identified an interaction between dimers that allows helical oligomerization and membrane tubulation. Using mutations that block either membrane binding or tubulation, we find that membrane binding is required for Imp2's cytokinetic function but that oligomerization and tubulation, activities often deemed central to F-BAR protein function, are dispensable. Accordingly, F-BARs that do not have the capacity to tubulate membranes functionally substitute for the Imp2 F-BAR, establishing that its major role is as a cell-cycle-regulated bridge between the membrane and Imp2 protein partners, rather than as a driver of membrane curvature.
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http://dx.doi.org/10.1016/j.celrep.2015.12.062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731314PMC
January 2016

Expanding the 3-O-Sulfate Proteome--Enhanced Binding of Neuropilin-1 to 3-O-Sulfated Heparan Sulfate Modulates Its Activity.

ACS Chem Biol 2016 Apr 14;11(4):971-80. Epub 2016 Jan 14.

Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky , Lexington, Kentucky 40536, United States.

Binding of proteins to heparan sulfate is driven predominantly by electrostatic interactions between positively charged amino acid residues in the protein and negatively charged sulfate groups located at various positions along the polysaccharide chain. Although many heparin/heparan-sulfate-binding proteins have been described, few exhibit preferential binding for heparan sulfates containing relatively rare 3-O-sulfated glucosamine residues. To expand the "3-O-sulfate proteome," affinity matrices were created from Chinese hamster ovary (CHO) cell heparan sulfate engineered in vitro with and without 3-O-sulfate groups. Fractionation of different animal sera yielded several proteins that bound specifically to columns containing 3-O-sulfated heparan sulfate modified by two members of the heparan sulfate 3-O-sulfotransferase superfamily, Hs3st1 and Hs3st2. Neuropilin-1 was analyzed in detail because it has been implicated in angiogenesis and axon guidance. We show that 3-O-sulfation enhanced the binding of neuropilin-1 to heparan sulfate immobilized on plastic plates and to heparan sulfate present on cultured cells. Chemoenzymatically synthesized 3-O-sulfated heparan sulfate dodecamers protected neuropilin-1 from thermal denaturation and inhibited neuropilin-1-dependent, semaphorin-3a-induced growth cone collapse of neurons derived from murine dorsal root ganglia. The effect of 3-O-sulfation was cell autonomous and specific to Hs3st2 based on collapse assays of neurons derived from Hs3st1- and Hs3st2-deficient mice. Finally, 3-O-sulfated heparan sulfate enhanced the inhibition of endothelial cell sprouting by exogenous heparan sulfate. These findings demonstrate a reliable method to identify members of the 3-O-sulfate proteome and that 3-O-sulfation of heparan sulfate can modulate axonal growth cone collapse and endothelial cell sprouting.
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http://dx.doi.org/10.1021/acschembio.5b00897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450942PMC
April 2016

Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis.

Dev Cell 2015 Dec;35(6):725-36

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. Electronic address:

F-BAR proteins function in diverse cellular processes by linking membranes to the actin cytoskeleton. Through oligomerization, multiple F-BAR domains can bend membranes into tubules, though the physiological importance of F-BAR-to-F-BAR assemblies is not yet known. Here, we investigate the F-BAR domain of the essential cytokinetic scaffold, Schizosaccharomyces pombe Cdc15, during cytokinesis. Challenging a widely held view that membrane deformation is a fundamental property of F-BARs, we report that the Cdc15 F-BAR binds, but does not deform, membranes in vivo or in vitro, and six human F-BAR domains-including those from Fer and RhoGAP4-share this property. Nevertheless, tip-to-tip interactions between F-BAR dimers are critical for Cdc15 oligomerization and high-avidity membrane binding, stabilization of contractile ring components at the medial cortex, and the fidelity of cytokinesis. F-BAR oligomerization is also critical for Fer and RhoGAP4 physiological function, demonstrating its broad importance to F-BAR proteins that function without membrane bending.
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http://dx.doi.org/10.1016/j.devcel.2015.11.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691284PMC
December 2015

Novel role of 4-hydroxy-2-nonenal in AIFm2-mediated mitochondrial stress signaling.

Free Radic Biol Med 2016 Feb 9;91:68-80. Epub 2015 Dec 9.

Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY, USA. Electronic address:

Cardiovascular complications are major side effects of many anticancer drugs. Accumulated evidence indicates that oxidative stress in mitochondria plays an important role in cardiac injury, but how mitochondrial redox mechanisms are involved in cardiac dysfunction remains unclear. Here, we demonstrate that 4-hydroxy-2-nonenal (HNE) activates the translocation of the mitochondrial apoptosis inducing factor (AIFm2) and facilitates apoptosis in heart tissue of mice and humans. Doxorubicin treatments significantly enhance cardiac levels of HNE and AIFm2. HNE adduction of AIFm2 inactivates the NADH oxidoreductase activity of AIFm2 and facilitates its translocation from mitochondria. His 174 on AIFm2 is the critical target of HNE adduction that triggers this functional switch. HNE adduction and translocation of AIFm2 from mitochondria upon Doxorubicin treatment are attenuated by superoxide dismutase mimetics. These results identify a previously unrecognized role of HNE with important consequences for mitochondrial stress signaling, heart failure, and the side effects of cancer therapy.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761499PMC
February 2016

Angiotensinogen Exerts Effects Independent of Angiotensin II.

Arterioscler Thromb Vasc Biol 2016 Feb 17;36(2):256-65. Epub 2015 Dec 17.

From the Saha Cardiovascular Research Center (H.L., C.W., D.A.H., A.B., J.J.M., X.C., M.Z., A.D.); Departments of Physiology (H.L., A.D.), Pharmacology and Nutritional Sciences (C.W., L.A.C., A.D.), and Molecular and Cellular Biochemistry (C.W.V.K.), University of Kentucky, Lexington; Isis Pharmaceuticals, Inc, Carlsbad, CA (M.J.G., A.E.M., R.M.C.); and Novartis Pharmaceuticals Corporation, East Hanover, NJ (D.L.F.).

Objective: This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations.

Approach And Results: All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis.

Conclusions: AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.
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http://dx.doi.org/10.1161/ATVBAHA.115.306740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732917PMC
February 2016

Neuropilin Functions as an Essential Cell Surface Receptor.

J Biol Chem 2015 Dec 8;290(49):29120-6. Epub 2015 Oct 8.

From the Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536

The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.
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http://dx.doi.org/10.1074/jbc.R115.687327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705917PMC
December 2015

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates.

J Biol Chem 2015 Sep 31;290(38):23361-70. Epub 2015 Jul 31.

From the Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536,

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.
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http://dx.doi.org/10.1074/jbc.M115.658203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645622PMC
September 2015

Structural basis for VEGF-C binding to neuropilin-2 and sequestration by a soluble splice form.

Structure 2015 Apr 5;23(4):677-87. Epub 2015 Mar 5.

Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA. Electronic address:

Vascular endothelial growth factor C (VEGF-C) is a potent lymphangiogenic cytokine that signals via the coordinated action of two cell surface receptors, Neuropilin-2 (Nrp2) and VEGFR-3. Diseases associated with both loss and gain of VEGF-C function, lymphedema and cancer, respectively, motivate studies of VEGF-C/Nrp2 binding and inhibition. Here, we demonstrate that VEGF-C binding to Nrp2 is regulated by C-terminal proteolytic maturation. The structure of the VEGF-C C terminus in complex with the ligand binding domains of Nrp2 demonstrates that a cryptic Nrp2 binding motif is released upon proteolysis, allowing specific engagement with the b1 domain of Nrp2. Based on the identified structural requirements for Nrp2 binding to VEGF-C, we hypothesized that the endogenous secreted splice form of Nrp2, s9Nrp2, may function as a selective inhibitor of VEGF-C. We find that s9Nrp2 forms a stable dimer that potently inhibits VEGF-C/Nrp2 binding and cellular signaling. These data provide critical insight into VEGF-C/Nrp2 binding and inhibition.
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http://dx.doi.org/10.1016/j.str.2015.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394031PMC
April 2015
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