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Andrew Bateman - One of the best experts on this subject based on the ideXlab platform.
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structure dissection of zebrafish Progranulins identifies a well folded granulin epithelin module protein with pro cell survival activities
Protein Science, 2018Co-Authors: Andrew Bateman, Babykumari P Chitramuthu, Feng Ni, Ping Xu, H P J Bennett, Ping WangAbstract:: The ancient and pluripotent Progranulins contain multiple repeats of a cysteine-rich sequence motif of ∼60 amino acids, called the granulin/epithelin module (GEM) with a prototypic structure of four β-hairpins zipped together by six inter-hairpin disulfide bonds. Prevalence of this disulfide-enforced structure is assessed here by an expression screening of 19 unique GEM sequences of the four Progranulins in the zebrafish genome, Progranulins 1, 2, A and B. While a majority of the expressed GEM peptides did not exhibit uniquely folded conformations, module AaE from Progranulin A and AbB from Progranulin B were found to fold into the protopypic 4-hairpin structure along with disulfide formation. Module AaE has the most-rigid three-dimensional structure with all four β-hairpins defined using high-resolution (H-15 N) NMR spectroscopy, including 492 inter-proton nuclear Overhauser effects, 23 3 J(HN,Hα ) coupling constants, 22 hydrogen bonds as well as 45 residual dipolar coupling constants. Three-dimensional structure of AaE and the partially folded AbB re-iterate the conformational stability of the N-terminal stack of two beta-hairpins and varying degrees of structural flexibility for the C-terminal half of the 4-hairpin global fold of the GEM repeat. A cell-based assay demonstrated a functional activity for the zebrafish granulin AaE in promoting the survival of neuronal cells, similarly to what has been found for the corresponding granulin E module in human Progranulin. Finally, this work highlights the remaining challenges in structure-activity studies of proteins containing the GEM repeats, due to the apparent prevalence of structural disorder in GEM motifs despite potentially a high density of intramolecular disulfide bonds.
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Methods for Expression and Purification of Biologically Active Recombinant Progranulin.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Chun Yu Zhao, Andrew BatemanAbstract:Purifying Progranulin may be useful in a variety of situations, for example, after it has been mutated or otherwise modified or when working with a species for which commercially produced Progranulin is unavailable. A method to express and purify human Progranulin is presented. Progranulin is transiently expressed in mammalian cells and isolated from their conditioned medium before purification by reversed-phase high-performance liquid chromatography (RP-HPLC). Human Progranulin is used as an example, but the protocol can be applied to any other Progranulin protein. Modifications of the expression-purification strategy for metabolic labeling of Progranulin and analytical systems based on heparin-affinity chromatography are presented.
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A Brief Overview of Progranulin in Health and Disease.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Andrew Bateman, Siu Tim Cheung, Hugh P.j. BennettAbstract:The purpose of this brief overview of the Progranulin protein is to provide a sense of the range and extent of the roles of Progranulin in normal physiology and pathology. Progranulin has received attention due to its role in neurodegeneration, where mutation of a single copy of GRN, the gene encoding Progranulin, results in frontotemporal dementia, whereas viral delivery of Progranulin to the brains of mice exhibiting Parkinson's or Alzheimer's disease phenotypes inhibits the progression of the neurodegenerative phenotypes. Of equal importance, Progranulin protects tissues against the harmful effects of poorly controlled inflammation and promotes tissue regeneration after injury at a multitude of sites throughout the body. Progranulin is overexpressed by many types of cancer and contributes to their progression. Given suitable analytical methods and model systems, Progranulin offers a wealth of research possibilities.
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Methods to Investigate the Roles of Progranulin in Angiogenesis Using In Vitro Strategies and Transgenic Mouse Models.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Huishi Toh, Eugene Daniels, Andrew BatemanAbstract:Endothelial cells activate the expression of Progranulin during angiogenesis. Here we discuss methods to investigate Progranulin activity on endothelial cells in vitro and on aortic explants. We then discuss methods to generate transgenic mice in which Progranulin expression is targeted to endothelial cells. These mice can be used to study the influence of Progranulin on angiogenesis during development in vivo. The transgenic strategy summarized here could be readily adapted to investigate the roles of Progranulin in other cell types and tissues by use of appropriate targeting vectors to drive the expression of Progranulin in the cell type of choice.
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chromatographic methods for the purification of granulin peptides
Methods of Molecular Biology, 2018Co-Authors: Andrew Bateman, Babykumari P Chitramuthu, H P J BennettAbstract:: Progranulin is composed of seven repeating cysteine-rich granulin domains. In some cells and tissues, the Progranulin is fragmented by proteolysis to generate the granulin modules as individual peptides, which are collectively referred to as granulins. These peptides are often biologically active, but the activity need not be identical to that of the parental Progranulin from which they are derived. Thus, some granulin peptides stimulate cell proliferation, as does Progranulin itself, while other granulin peptides suppress proliferation. Similarly, some granulin peptides promote inflammation even though Progranulin itself suppresses inflammation. Investigating the structural and biological properties of granulin peptides is challenging. Here we discuss methods that employ reversed-phase high-performance liquid chromatography (RP-HPLC) and in some instances size-exclusion high-performance liquid chromatography (SE-HPLC) to isolate granulin peptides from tissues, in particular those that are rich in inflammatory cells such as neutrophils, bone marrow, or hematopoietic organs of teleost fish.
Renato V. Iozzo - One of the best experts on this subject based on the ideXlab platform.
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Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer.
Matrix biology : journal of the International Society for Matrix Biology, 2020Co-Authors: Simone Buraschi, Thomas Neill, Chiara Palladino, Antonino Belfiore, Renato V. Iozzo, Andrea MorrioneAbstract:Abstract The growth factor Progranulin plays a critical role in bladder cancer by modulating tumor cell motility and invasion. Progranulin regulates remodeling of the actin cytoskeleton by interacting with drebrin, an actin binding protein that regulates tumor growth. We previously discovered that Progranulin depletion inhibits epithelial-to-mesenchymal transition and markedly reduces in vivo tumor growth. Moreover, Progranulin depletion sensitizes urothelial cancer cells to cisplatin treatment, further substantiating a pro-survival function of Progranulin. Until recently, the Progranulin signaling receptor remained unidentified, precluding a full understanding of Progranulin action in tumor cell biology. We recently identified EphA2, a member of a large family of receptor tyrosine-kinases, as the functional receptor for Progranulin. However, it is not established whether EphA2 plays an oncogenic role in bladder cancer. Here we demonstrate that Progranulin, and not ephrin-A1, the canonical ligand for EphA2, is the predominant EphA2 ligand in bladder cancer. Progranulin evoked Akt- and Erk1/2-mediated EphA2 phosphorylation at Ser897, which could drive bladder tumorigenesis. We discovered that EphA2 depletion severely blunted Progranulin-dependent motility and anchorage-independent growth, and sensitized bladder cancer cells to cisplatin treatment. We further defined the mechanisms of Progranulin/EphA2-dependent motility by identifying liprin-α1 as a novel Progranulin-dependent EphA2 interacting protein and establishing its critical role in cell motility. The discovery of EphA2 as the functional signaling receptor for Progranulin and the identification of novel downstream effectors offer a new avenue for understanding the underlying mechanism of Progranulin action and may constitute novel clinical and therapeutic targets in bladder cancer.
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Analysis of Progranulin-Mediated Akt and MAPK Activation
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Simone Buraschi, Manuela Stefanello, Ryuta Tanimoto, Antonino Belfiore, Renato V. Iozzo, Andrea MorrioneAbstract:Progranulin has emerged in recent years as an important regulator of various biological functions including cell proliferation, wound healing, motility, and protection from apoptosis. Progranulin is also critical for transformation as established in several cancer models.Progranulin biological responses elicit through the activation of the Akt and MAPK pathways, which are critical for Progranulin downstream signaling.In this chapter various experimental approaches aiming at detecting Progranulin-mediated Akt and MAPK activation will be discussed.
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The perlecan-interacting growth factor Progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin.
Matrix biology : journal of the International Society for Matrix Biology, 2017Co-Authors: Ryuta Tanimoto, Simone Buraschi, Stephen C. Peiper, Leonard G. Gomella, Thomas Neill, Chiara Palladino, Antonino Belfiore, Renato V. Iozzo, Andrea MorrioneAbstract:Despite extensive clinical and experimental studies over the past decades, the pathogenesis and progression to the castration-resistant stage of prostate cancer remains largely unknown. Progranulin, a secreted growth factor, strongly binds the heparin-sulfate proteoglycan perlecan, and counteracts its biological activity. We established that Progranulin acts as an autocrine growth factor and promotes prostate cancer cell motility, invasion, and anchorage-independent growth. Progranulin was overexpressed in prostate cancer tissues vis-a-vis non-neoplastic tissues supporting the hypothesis that Progranulin may play a key role in prostate cancer progression. However, Progranulin's mode of action is not well understood and proteins regulating Progranulin signaling have not been identified. Sortilin, a single-pass type I transmembrane protein of the Vps10 family, binds Progranulin in neurons and targets Progranulin for lysosomal degradation. Significantly, in DU145 and PC3 cells, we detected very low levels of sortilin associated with high levels of Progranulin production and enhanced motility. Restoring sortilin expression decreased Progranulin levels, inhibited motility and anchorage-independent growth and destabilized Akt. These results demonstrated a critical role for sortilin in regulating Progranulin and suggest that sortilin loss may contribute to prostate cancer progression. Here, we provide the novel observation that Progranulin downregulated sortilin protein levels independent of transcription. Progranulin induced sortilin ubiquitination, internalization via clathrin-dependent endocytosis and sorting into early endosomes for lysosomal degradation. Collectively, these results constitute a regulatory feed-back mechanism whereby sortilin downregulation ensures sustained Progranulin-mediated oncogenesis.
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EphA2 is a functional receptor for the growth factor Progranulin.
The Journal of cell biology, 2016Co-Authors: Thomas Neill, Simone Buraschi, Andrea Morrione, Atul Goyal, Catherine Sharpe, Elizabeth Natkanski, Liliana Schaefer, Renato V. IozzoAbstract:Although the growth factor Progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for Progranulin. Recombinant Progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of Progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of Progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for Progranulin offers a new avenue for understanding the underlying mode of action of Progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases.
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Mechanisms of Progranulin Action and Regulation in Genitourinary Cancers
Frontiers in endocrinology, 2016Co-Authors: Ryuta Tanimoto, Simone Buraschi, Antonino Belfiore, Renato V. Iozzo, Andrea MorrioneAbstract:The growth factor Progranulin has emerged in recent years as a critical regulator of transformation in several cancer models, including breast cancer, glioblastomas, leukemias, and hepatocellular carcinomas. Several laboratories, including ours, have also demonstrated an important role of Progranulin in several genitourinary cancers, including ovarian, endometrial, cervical, prostate, and bladder tumors, where Progranulin acts as an autocrine growth factor thereby modulating motility and invasion of transformed cells. In this review, we will focus on the mechanisms of action and regulation of Progranulin signaling in genitourinary cancers with a special emphasis on prostate and bladder tumors.
Terri L. Petkau - One of the best experts on this subject based on the ideXlab platform.
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Human Progranulin-expressing mice as a novel tool for the development of Progranulin-modulating therapeutics.
Neurobiology of disease, 2021Co-Authors: Terri L. Petkau, Benjamin Life, Jasmine Yang, Oriol Fornes, Wyeth W. Wasserman, Elizabeth Simpson, Blair R. LeavittAbstract:Abstract The granulin protein (also known as, and hereafter referred to as, Progranulin) is a secreted glycoprotein that contributes to overall brain health. Heterozygous loss-of-function mutations in the gene encoding the Progranulin protein (Granulin Precursor, GRN) are a common cause of familial frontotemporal dementia (FTD). Gene therapy approaches that aim to increase Progranulin expression from a single wild-type allele, an area of active investigation for the potential treatment of GRN-dependent FTD, will benefit from the availability of a mouse model that expresses a genomic copy of the human GRN gene. Here we report the development and characterization of a novel mouse model that expresses the entire human GRN gene in its native genomic context as a single copy inserted into a defined locus (Hprt) in the mouse genome. We show that human and mouse Progranulin are expressed in a similar tissue-specific pattern, suggesting that the two genes are regulated by similar mechanisms. Human Progranulin rescues a phenotype characteristic of Progranulin-null mice, the exaggerated and early deposition of the aging pigment lipofuscin in the brain, indicating that the two proteins are functionally similar. Longitudinal behavioural and neuropathological analyses revealed no significant differences between wild-type and human Progranulin-overexpressing mice up to 18 months of age, providing evidence that long-term increase of Progranulin levels is well tolerated in mice. Finally, we demonstrate that human Progranulin expression can be increased in the brain using an antisense oligonucleotide that inhibits a known GRN-regulating micro-RNA, demonstrating that the transgene is responsive to potential gene therapy drugs. Human Progranulin-expressing mice represent a novel and valuable tool to expedite the development of Progranulin-modulating therapeutics.
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Selective depletion of microglial Progranulin in mice is not sufficient to cause neuronal ceroid lipofuscinosis or neuroinflammation.
Journal of neuroinflammation, 2017Co-Authors: Terri L. Petkau, Natalia Kosior, Kathleen De Asis, Colum Connolly, Blair R. LeavittAbstract:Progranulin deficiency due to heterozygous null mutations in the GRN gene are a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations are thought to be a rare cause of neuronal ceroid lipofuscinosis (NCL). Aged Progranulin-knockout (Grn-null) mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. In the brain, Progranulin is predominantly expressed in neurons and microglia, and previously, we demonstrated that neuronal-specific depletion of Progranulin does not recapitulate the neuropathological phenotype of Grn-null mice. In this study, we evaluated whether selective depletion of Progranulin expression in myeloid-lineage cells, including microglia, causes NCL-like neuropathology or neuroinflammation in mice. We generated mice with Progranulin depleted in myeloid-lineage cells by crossing mice homozygous for a floxed Progranulin allele to mice expressing Cre recombinase under control of the LyzM promotor (Lyz-cKO). Progranulin expression was reduced by approximately 50–70% in isolated microglia compared to WT levels. Lyz-cKO mice aged to 12 months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for many of these measures in Grn-null animals. To evaluate the functional effect of reduced Progranulin expression in isolated microglia, primary cultures were stimulated with controlled standard endotoxin and cytokine release was measured. While Grn-null microglia display a hyper-inflammatory phenotype, Lyz-cKO and WT microglia secreted similar levels of inflammatory cytokines. We conclude that Progranulin expression from either microglia or neurons is sufficient to prevent the development of NCL-like neuropathology in mice. Furthermore, microglia that are deficient for Progranulin expression but isolated from a Progranulin-rich environment have a normal inflammatory profile. Our results suggest that Progranulin acts, at least partly, in a non-cell autonomous manner in the brain.
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Core neuropathological abnormalities in Progranulin-deficient mice are penetrant on multiple genetic backgrounds.
Neuroscience, 2015Co-Authors: Terri L. Petkau, Austin Hill, Blair R. LeavittAbstract:Loss-of-function mutations in the Progranulin gene (GRN) are a common cause of familial frontotemporal lobar degeneration (FTLD). A high degree of heterogeneity in the age-of-onset, duration of disease, and clinical presentation of FTLD, even among families carrying the same GRN mutation, suggests that additional modifying genes may be important to pathogenesis. Progranulin-knockout mice display subtle behavioral abnormalities and progressive neuropathological changes, as well as altered dendritic morphology and synaptic deficits in the hippocampus. In this study we evaluated multiple neuropathological endpoints in aged Progranulin knockout mice and their wild-type littermates on two different genetic backgrounds: C57Bl/6 and 129/SvImJ. We find that in most brain regions, both strains are susceptible to Progranulin-mediated neuropathological phenotypes, including astrogliosis, microgliosis, and highly accelerated deposition of the aging pigment lipofuscin. Neuroinflammation due to Progranulin deficiency is exaggerated in the B6 strain and present, but less pronounced, in the 129 strain. Differences between the strains in hippocampal neuron counts and neuronal morphology suggest a complex role for Progranulin in the hippocampus. We conclude that core Progranulin-mediated neurodegenerative phenotypes are penetrant on multiple inbred mouse strains, but that genetic background modulates Progranulin's role in neuroinflammation and hippocampal biology.
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Progranulin in neurodegenerative disease
Trends in neurosciences, 2014Co-Authors: Terri L. Petkau, Blair R. LeavittAbstract:Loss-of-function mutations in the Progranulin gene are a common cause of familial frontotemporal dementia (FTD). The purpose of this review is to summarize the role of Progranulin in health and disease, because the field is now poised to begin examining therapeutics that alter endogenous Progranulin levels. We first review the clinical and neuropathological phenotype of FTD patients carrying mutations in the Progranulin gene, which suggests that Progranulin-mediated neurodegeneration is multifactorial and influenced by other genetic and/or environmental factors. We then examine evidence for the role of Progranulin in the brain with a focus on mouse model systems. A better understanding of the complexity of Progranulin biology in the brain will help guide the development of Progranulin-modulating therapies for neurodegenerative disease.
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Synaptic dysfunction in Progranulin-deficient mice.
Neurobiology of disease, 2011Co-Authors: Terri L. Petkau, Austin Hill, Scott J. Neal, Austen J. Milnerwood, Ada Mew, Paul C. Orban, Jenny Gregg, Howard Feldman, Ian R. MackenzieAbstract:Progranulin haploinsufficiency is a common cause of familial frontotemporal dementia (FTD), but the role of Progranulin in the brain is poorly understood. To investigate the role of murine Progranulin (Grn) in the CNS in vivo, we generated mice targeted at the Progranulin locus (Grn) using a gene-trap vector. Constitutive Progranulin knockout mice (GrnKO) show moderate abnormalities in anxiety-related behaviors, social interactions, motor coordination, and novel object recognition at 8months of age, many of which differ between males and females. Analysis of synaptic transmission in 10-12 month old GrnKO male mice indicates altered synaptic connectivity and impaired synaptic plasticity. Additionally, apical dendrites in pyramidal cells in the CA1 region of the hippocampus in GrnKO males display an altered morphology and have significantly decreased spine density compared to wild-type (WT) mice. The observed changes in behavior, synaptic transmission, and neuronal morphology in GrnKO mice occur prior to neuropathological abnormalities, most of which are apparent at 18 but not at 8 months of age. We conclude that Progranulin deficiency leads to reduced synaptic connectivity and impaired plasticity, which may contribute to FTD pathology in human patients.
Hugh P.j. Bennett - One of the best experts on this subject based on the ideXlab platform.
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A Brief Overview of Progranulin in Health and Disease.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Andrew Bateman, Siu Tim Cheung, Hugh P.j. BennettAbstract:The purpose of this brief overview of the Progranulin protein is to provide a sense of the range and extent of the roles of Progranulin in normal physiology and pathology. Progranulin has received attention due to its role in neurodegeneration, where mutation of a single copy of GRN, the gene encoding Progranulin, results in frontotemporal dementia, whereas viral delivery of Progranulin to the brains of mice exhibiting Parkinson's or Alzheimer's disease phenotypes inhibits the progression of the neurodegenerative phenotypes. Of equal importance, Progranulin protects tissues against the harmful effects of poorly controlled inflammation and promotes tissue regeneration after injury at a multitude of sites throughout the body. Progranulin is overexpressed by many types of cancer and contributes to their progression. Given suitable analytical methods and model systems, Progranulin offers a wealth of research possibilities.
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The Evolution of the Secreted Regulatory Protein Progranulin.
PloS one, 2015Co-Authors: Roger G. E. Palfree, Hugh P.j. Bennett, Andrew BatemanAbstract:Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human Progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from Progranulin by proteolysis as 6kDa granulin polypeptides. Both intact Progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and Progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate Progranulin and granulin module evolution by investigating (i): the origins of metazoan Progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate Progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that Progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that Progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide sequence conservation of mammalian granulin modules identified potential structure-activity relationships that may be informative in designing Progranulin based therapeutics.
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Structure, Function, and Mechanism of Progranulin; the Brain and Beyond
Journal of molecular neuroscience : MN, 2011Co-Authors: Huishi Toh, Hugh P.j. Bennett, Babykumari P Chitramuthu, Andrew BatemanAbstract:Mutation of human GRN, the gene encoding the secreted glycoprotein Progranulin, results in a form of frontotemporal lobar degeneration that is characterized by the presence of ubiquitinated inclusions containing phosphorylated and cleaved fragments of the transactivation response element DNA-binding protein-43. This has stimulated interest in understanding the role of Progranulin in the central nervous system, and in particular, how this relates to neurodegeneration. Progranulin has many roles outside the brain, including regulation of cellular proliferation, survival, and migration, in cancer, including cancers of the brain, in wound repair, and inflammation. It often acts through the extracellular signal-regulated kinase and phopshatidylinositol-3-kinases pathways. The neurobiology of Progranulin has followed a similar pattern with proposed roles for Progranulin (PGRN) in the central nervous system as a neuroprotective agent and in neuroinflammation. Here we review the structure, biology, and mechanism of Progranulin action. By understanding PGRN in a wider context, we may be better able to delineate its roles in the normal brain and in neurodegenerative disease.
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The zebrafish Progranulin gene family and antisense transcripts
BMC genomics, 2005Co-Authors: Benoît Cadieux, Babykumari P Chitramuthu, David C. Baranowski, Hugh P.j. BennettAbstract:Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian Progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian Progranulin may be represented by two co-orthologues in zebrafish. The cDNAs encoding two zebrafish granulin precursors, Progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, Progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric Progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of Progranulin. Chromosomal localization of the four Progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs. In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four paralogues in zebrafish.
Babykumari P Chitramuthu - One of the best experts on this subject based on the ideXlab platform.
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structure dissection of zebrafish Progranulins identifies a well folded granulin epithelin module protein with pro cell survival activities
Protein Science, 2018Co-Authors: Andrew Bateman, Babykumari P Chitramuthu, Feng Ni, Ping Xu, H P J Bennett, Ping WangAbstract:: The ancient and pluripotent Progranulins contain multiple repeats of a cysteine-rich sequence motif of ∼60 amino acids, called the granulin/epithelin module (GEM) with a prototypic structure of four β-hairpins zipped together by six inter-hairpin disulfide bonds. Prevalence of this disulfide-enforced structure is assessed here by an expression screening of 19 unique GEM sequences of the four Progranulins in the zebrafish genome, Progranulins 1, 2, A and B. While a majority of the expressed GEM peptides did not exhibit uniquely folded conformations, module AaE from Progranulin A and AbB from Progranulin B were found to fold into the protopypic 4-hairpin structure along with disulfide formation. Module AaE has the most-rigid three-dimensional structure with all four β-hairpins defined using high-resolution (H-15 N) NMR spectroscopy, including 492 inter-proton nuclear Overhauser effects, 23 3 J(HN,Hα ) coupling constants, 22 hydrogen bonds as well as 45 residual dipolar coupling constants. Three-dimensional structure of AaE and the partially folded AbB re-iterate the conformational stability of the N-terminal stack of two beta-hairpins and varying degrees of structural flexibility for the C-terminal half of the 4-hairpin global fold of the GEM repeat. A cell-based assay demonstrated a functional activity for the zebrafish granulin AaE in promoting the survival of neuronal cells, similarly to what has been found for the corresponding granulin E module in human Progranulin. Finally, this work highlights the remaining challenges in structure-activity studies of proteins containing the GEM repeats, due to the apparent prevalence of structural disorder in GEM motifs despite potentially a high density of intramolecular disulfide bonds.
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chromatographic methods for the purification of granulin peptides
Methods of Molecular Biology, 2018Co-Authors: Andrew Bateman, Babykumari P Chitramuthu, H P J BennettAbstract:: Progranulin is composed of seven repeating cysteine-rich granulin domains. In some cells and tissues, the Progranulin is fragmented by proteolysis to generate the granulin modules as individual peptides, which are collectively referred to as granulins. These peptides are often biologically active, but the activity need not be identical to that of the parental Progranulin from which they are derived. Thus, some granulin peptides stimulate cell proliferation, as does Progranulin itself, while other granulin peptides suppress proliferation. Similarly, some granulin peptides promote inflammation even though Progranulin itself suppresses inflammation. Investigating the structural and biological properties of granulin peptides is challenging. Here we discuss methods that employ reversed-phase high-performance liquid chromatography (RP-HPLC) and in some instances size-exclusion high-performance liquid chromatography (SE-HPLC) to isolate granulin peptides from tissues, in particular those that are rich in inflammatory cells such as neutrophils, bone marrow, or hematopoietic organs of teleost fish.
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Structure, Function, and Mechanism of Progranulin; the Brain and Beyond
Journal of molecular neuroscience : MN, 2011Co-Authors: Huishi Toh, Hugh P.j. Bennett, Babykumari P Chitramuthu, Andrew BatemanAbstract:Mutation of human GRN, the gene encoding the secreted glycoprotein Progranulin, results in a form of frontotemporal lobar degeneration that is characterized by the presence of ubiquitinated inclusions containing phosphorylated and cleaved fragments of the transactivation response element DNA-binding protein-43. This has stimulated interest in understanding the role of Progranulin in the central nervous system, and in particular, how this relates to neurodegeneration. Progranulin has many roles outside the brain, including regulation of cellular proliferation, survival, and migration, in cancer, including cancers of the brain, in wound repair, and inflammation. It often acts through the extracellular signal-regulated kinase and phopshatidylinositol-3-kinases pathways. The neurobiology of Progranulin has followed a similar pattern with proposed roles for Progranulin (PGRN) in the central nervous system as a neuroprotective agent and in neuroinflammation. Here we review the structure, biology, and mechanism of Progranulin action. By understanding PGRN in a wider context, we may be better able to delineate its roles in the normal brain and in neurodegenerative disease.
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Progranulin is expressed within motor neurons and promotes neuronal cell survival.
BMC neuroscience, 2009Co-Authors: Cara L Ryan, Babykumari P Chitramuthu, David C. Baranowski, Suneil Malik, Mingju Cao, Sandra Minotti, Heather D. Durham, Denis G. Kay, Christopher A. ShawAbstract:Background Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the Progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U) associated with the formation of ubiquitinated inclusions. Recent reports indicate that Progranulin has neurotrophic effects, which, if confirmed would make Progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high Progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated Progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of Progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival.
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The zebrafish Progranulin gene family and antisense transcripts
BMC genomics, 2005Co-Authors: Benoît Cadieux, Babykumari P Chitramuthu, David C. Baranowski, Hugh P.j. BennettAbstract:Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian Progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian Progranulin may be represented by two co-orthologues in zebrafish. The cDNAs encoding two zebrafish granulin precursors, Progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, Progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric Progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of Progranulin. Chromosomal localization of the four Progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs. In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four paralogues in zebrafish.
