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1.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34140407

RESUMO

In 2006, GRN mutations were first linked to frontotemporal dementia (FTD), the leading cause of non-Alzheimer dementias. While much research has been dedicated to understanding the genetic causes of the disease, our understanding of the mechanistic impacts of GRN deficiency has only recently begun to take shape. With no known cure or treatment available for GRN-related FTD, there is a growing need to rapidly advance genetic and/or small-molecule therapeutics for this disease. This issue is complicated by the fact that, while lysosomal dysfunction seems to be a key driver of pathology, the mechanisms linking a loss of GRN to a pathogenic state remain unclear. In our attempt to address these key issues, we have turned to the nematode, Caenorhabditis elegans, to model, study, and find potential therapies for GRN-deficient FTD. First, we show that the loss of the nematode GRN ortholog, pgrn-1, results in several behavioral and molecular defects, including lysosomal dysfunction and defects in autophagic flux. Our investigations implicate the sphingolipid metabolic pathway in the regulation of many of the in vivo defects associated with pgrn-1 loss. Finally, we utilized these nematodes as an in vivo tool for high-throughput drug screening and identified two small molecules with potential therapeutic applications against GRN/pgrn-1 deficiency. These compounds reverse the biochemical, cellular, and functional phenotypes of GRN deficiency. Together, our results open avenues for mechanistic and therapeutic research into the outcomes of GRN-related neurodegeneration, both genetic and molecular.


Assuntos
Autofagia/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Lisossomos/genética , Progranulinas/metabolismo , Acetofenonas/farmacologia , Animais , Benzopiranos/farmacologia , Vias Biossintéticas , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Avaliação Pré-Clínica de Medicamentos , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Mutação/genética , Fenótipo , Progranulinas/genética , Rivastigmina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Esfingolipídeos/metabolismo
2.
Methods Mol Biol ; 1806: 19-34, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29956266

RESUMO

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.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Granulinas/isolamento & purificação , Animais , Medula Óssea/metabolismo , Carpas/metabolismo , Cromatografia de Fase Reversa , Rim Cefálico/metabolismo , Humanos , Espectrometria de Massas , Peso Molecular , Neutrófilos/metabolismo , Ratos Sprague-Dawley , Baço/metabolismo , Extratos de Tecidos
3.
Methods Mol Biol ; 1806: 3-15, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29956265

RESUMO

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.


Assuntos
Doença , Saúde , Progranulinas/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Modelos Biológicos , Progranulinas/química
4.
Methods Mol Biol ; 1806: 207-231, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29956279

RESUMO

The zebrafish (Danio rerio), a small tropical fish, has become a powerful model for the study of early vertebrate development, human diseases, and drug screening. Zebrafish provides large numbers of optically clear embryos, and its development is very rapid. Overexpression or under-expression of proteins can be effectively achieved by microinjection of mRNA or morpholino antisense oligonucleotides (MOs), respectively, into developing embryos at the 1-2 cell stage. The function of a particular protein can be revealed by correlating gene expression patterns with the phenotypes observed from over- or under-expression. We defined the expression pattern of zebrafish progranulin A (zfPGRN-A), an orthologue to the single human PGRN by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). The MO-mediated knockdown of zfPGRN-A expression generated embryos that display abnormal motor neuron development resulting in touch-evoked swimming deficits.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Neurônios/metabolismo , Progranulinas/metabolismo , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Hibridização In Situ , Microinjeções , Morfolinos/farmacologia , Permeabilidade , Moldes Genéticos , Peixe-Zebra/embriologia
5.
Protein Sci ; 27(8): 1476-1490, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29732682

RESUMO

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 ß-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 ß-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.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Progranulinas/química , Progranulinas/farmacologia , Animais , Linhagem Celular , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Camundongos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Progranulinas/genética , Progranulinas/metabolismo , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Peixe-Zebra
6.
Brain ; 140(12): 3081-3104, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29053785

RESUMO

Progranulin, a secreted glycoprotein, is encoded in humans by the single GRN gene. Progranulin consists of seven and a half, tandemly repeated, non-identical copies of the 12 cysteine granulin motif. Many cellular processes and diseases are associated with this unique pleiotropic factor that include, but are not limited to, embryogenesis, tumorigenesis, inflammation, wound repair, neurodegeneration and lysosome function. Haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to frontotemporal lobar degeneration, a progressive neuronal atrophy that presents in patients as frontotemporal dementia. Frontotemporal dementia is an early onset form of dementia, distinct from Alzheimer's disease. The GRN-related form of frontotemporal lobar dementia is a proteinopathy characterized by the appearance of neuronal inclusions containing ubiquitinated and fragmented TDP-43 (encoded by TARDBP). The neurotrophic and neuro-immunomodulatory properties of progranulin have recently been reported but are still not well understood. Gene delivery of GRN in experimental models of Alzheimer's- and Parkinson's-like diseases inhibits phenotype progression. Here we review what is currently known concerning the molecular function and mechanism of action of progranulin in normal physiological and pathophysiological conditions in both in vitro and in vivo models. The potential therapeutic applications of progranulin in treating neurodegenerative diseases are highlighted.


Assuntos
Degeneração Lobar Frontotemporal/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Animais , Demência Frontotemporal/tratamento farmacológico , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/tratamento farmacológico , Degeneração Lobar Frontotemporal/metabolismo , Granulinas , Haploinsuficiência , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Modelos Neurológicos , Terapia de Alvo Molecular , Mutação , Progranulinas , Proteinopatias TDP-43/tratamento farmacológico , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/metabolismo
7.
PLoS One ; 12(3): e0174784, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28358904

RESUMO

Progranulin (PGRN) is a glycoprotein with multiple roles in normal and disease states. Mutations within the GRN gene cause frontotemporal lobar degeneration (FTLD). The affected neurons display distinctive TAR DNA binding protein 43 (TDP-43) inclusions. How partial loss of PGRN causes TDP-43 neuropathology is poorly understood. TDP-43 inclusions are also found in affected neurons of patients with other neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. In ALS, TDP-43 inclusions are typically also immunoreactive for fused in sarcoma (FUS). Mutations within TDP-43 or FUS are themselves neuropathogenic in ALS and some cases of FTLD. We used the outgrowth of caudal primary motor neurons (MNs) in zebrafish embryos to investigate the interaction of PGRN with TDP-43 and FUS in vivo. As reported previously, depletion of zebrafish PGRN-A (zfPGRN-A) is associated with truncated primary MNs and impaired motor function. Here we found that depletion of zfPGRN-A results in primary MNs outgrowth stalling at the horizontal myoseptum, a line of demarcation separating the myotome into dorsal and ventral compartments that is where the final destination of primary motor is assigned. Successful axonal outgrowth beyond the horizontal myoseptum depends in part upon formation of acetylcholine receptor clusters and this was found to be disorganized upon depletion of zfPGRN-A. PGRN reversed the effects of zfPGRN-A knockdown, but a related gene, zfPGRN-1, was without effect. Both knockdown of TDP-43 or FUS, as well as expression of humanTDP-43 and FUS mutants results in MN abnormalities that are reversed by co-expression of hPGRN mRNA. Neither TDP-43 nor FUS reversed MN phenotypes caused by the depletion of PGRN. Thus TDP-43 and FUS lie upstream of PGRN in a gene complementation pathway. The ability of PGRN to override TDP-43 and FUS neurotoxicity due to partial loss of function or mutation in the corresponding genes may have therapeutic relevance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neurônios Motores/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação/genética , Proteína FUS de Ligação a RNA/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
8.
PLoS One ; 10(8): e0133749, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26248158

RESUMO

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.


Assuntos
Evolução Molecular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sequência de Aminoácidos , Animais , Bases de Dados Genéticas , Fungos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Funções Verossimilhança , Dados de Sequência Molecular , Plantas/metabolismo , Progranulinas , Estrutura Terciária de Proteína , Alinhamento de Sequência
9.
J Vis Exp ; (80): e50644, 2013 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-24192694

RESUMO

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.


Assuntos
Técnicas de Cultura Embrionária/métodos , Hibridização In Situ/métodos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Embrião não Mamífero , Humanos , Sondas RNA/química , Sondas RNA/genética , RNA Antissenso/química , RNA Antissenso/genética
10.
Hum Mol Genet ; 22(4): 782-94, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23172908

RESUMO

The DNA/RNA binding proteins TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) are genetically linked to amyotrophic lateral sclerosis and frontotemporal lobar dementia, while the inappropriate cytoplasmic accumulations of TDP-43 and FUS are observed in a growing number of late-onset pathologies including spinocerebellar ataxia 3, Alzheimer's and Huntington's diseases (HD). To investigate if TDP-43 and FUS contribute to neurodegenerative phenotypes, we turned to a genetically accessible Caenorhabditis elegans model of polyglutamine toxicity. In C. elegans, we observe that genetic loss-of-function mutations for nematode orthologs of TDP-43 or FUS reduced behavioral defects and neurodegeneration caused by huntingtin exon-1 with expanded polyglutamines. Furthermore, using striatal cells from huntingtin knock-in mice we observed that small interfering ribonucleic acid (siRNA) against TDP-43 or FUS reduced cell death caused by mutant huntingtin. Moreover, we found that TDP-43 and the survival factor progranulin (PGRN) genetically interact to regulate polyglutamine toxicity in C. elegans and mammalian cells. Altogether our data point towards a conserved function for TDP-43 and FUS in promoting polyglutamine toxicity and that delivery of PGRN may have therapeutic benefits.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Doença de Huntington/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/patologia , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Granulinas , Histona Desacetilases/metabolismo , Humanos , Proteína Huntingtina , Doença de Huntington/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Progranulinas , RNA Interferente Pequeno/genética
11.
Methods Mol Biol ; 768: 273-96, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21805249

RESUMO

The Zebrafish (Danio rerio) is a powerful and well-established tool used extensively for the study of early vertebrate development and as a model of human diseases. Zebrafish genes orthologous to their mammalian counterparts generally share conserved biological function. Protein knockdown or overexpression can be effectively achieved by microinjection of morpholino antisense oligonucleotides (MOs) or mRNA, respectively, into developing embryos at the one- to two-cell stage. Correlating gene expression patterns with the characterizing of phenotypes resulting from over- or underexpression can reveal the function of a particular protein. The microinjection technique is simple and results are reproducible. We defined the expression pattern of the proprotein convertase PCSK5 within the lateral line neuromasts and various organs including the liver, gut and otic vesicle by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). MO-mediated knockdown of zebrafish PCSK5 expression generated embryos that display abnormal neuromast deposition within the lateral line system resulting in uncoordinated patterns of swimming.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Pró-Proteína Convertase 5 , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/metabolismo , Imunofluorescência , Hibridização In Situ , Sistema da Linha Lateral/fisiologia , Microinjeções , Neurônios/fisiologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Organogênese/genética , Pró-Proteína Convertase 5/genética , Pró-Proteína Convertase 5/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
J Mol Neurosci ; 45(3): 538-48, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21691802

RESUMO

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.


Assuntos
Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Encéfalo/anatomia & histologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/química , Mutação , Neoplasias/patologia , Neoplasias/fisiopatologia , Progranulinas , Cicatrização
13.
Dev Dyn ; 239(11): 2933-46, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20882679

RESUMO

Pro-protein convertase subtilisin/kexin 5 (PC5, also known as PC6) is a member of the subtilisin-like superfamily of serine proteases implicated in the maturation of latent precursor proteins into their functionally active derivatives. To investigate the functional roles, we have cloned the cDNA sequences encoding two candidate zebrafish PC5 convertases (designated as PCSK5.1 and PCSK5.2) co-orthologous to the single PC5 encoding gene (PCSK5) found in mammals. Both display syntenic correspondence to the human PCSK5 gene. Overall gene architecture has been conserved across species. While PC5.1 mRNA expression is very discrete within the otic vesicle and lateral line neuromasts, PC5.2 transcripts are more ubiquitously expressed within the central nervous system together with specific localization in various organs including liver, intestine, and otic vesicle. Zebrafish PC5.1-deficient embryos display abnormal neuromast deposition within the lateral line system and lack a normal touch response, consistent with the known sensory role that the lateral line plays in spatial awareness and sensing the environment.


Assuntos
Pró-Proteína Convertase 5/química , Pró-Proteína Convertase 5/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Embrião não Mamífero/metabolismo , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Pró-Proteína Convertase 5/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
14.
Bioessays ; 31(11): 1245-54, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19795409

RESUMO

The growth factor progranulin (PGRN) regulates cell division, survival, and migration. PGRN is an extracellular glycoprotein bearing multiple copies of the cysteine-rich granulin motif. With PGRN family members in plants and slime mold, it represents one of the most ancient of the extracellular regulatory proteins still extant in modern animals. PRGN has multiple biological roles. It contributes to the regulation of early embryogenesis, to adult tissue repair and inflammation. Elevated PGRN levels often occur in cancers, and PGRN immunotherapy inhibits the growth of hepatic cancer xenografts in mice. Recent studies have demonstrated roles for PGRN in neurobiology. An autosomal dominant mutation in GRN, the gene for PGRN, leads to neuronal atrophy in the frontal and temporal lobes, resulting in the disease frontotemporal lobar dementia. In this review we will discuss current knowledge of the multifaceted biology of PGRN.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Animais , Demência/genética , Lobo Frontal/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Moleculares , Família Multigênica , Neoplasias/genética , Doenças Neurodegenerativas/genética , Progranulinas , Cicatrização
15.
BMC Neurosci ; 10: 130, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19860916

RESUMO

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. RESULTS: In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months). This is mediated at least in part through an anti-apoptotic mechanism. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. Knockdown of progranulin expression using shRNA technology further reduced cell survival. CONCLUSION: Neurons are among the most long-lived cells in the body and are subject to low levels of toxic challenges throughout life. We have demonstrated that progranulin is abundantly expressed in motor neurons and is cytoprotective over prolonged periods when over-expressed in a neuronal cell line. This work highlights the importance of progranulin as neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including motor neuron disease.


Assuntos
Sobrevivência Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neurônios Motores/metabolismo , Medula Espinal/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Clonagem Molecular , Imunofluorescência , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Granulinas , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Microscopia Confocal , Neurônios Motores/ultraestrutura , Progranulinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/ultraestrutura , Transfecção
16.
Protein Sci ; 17(4): 711-24, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18359860

RESUMO

Progranulin is a secreted protein with important functions in several physiological and pathological processes, such as embryonic development, host defense, and wound repair. Autosomal dominant mutations in the progranulin gene cause frontotemporal dementia, while overexpression of progranulin promotes the invasive progression of a range of tumors, including those of the breast and the brain. Structurally, progranulin consists of seven-and-a-half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides. We have expressed all seven human GEMs using recombinant DNA in Escherichia coli. High-resolution NMR showed that only the three GEMs, hGrnA, hGrnC, and hGrnF, contain relatively well-defined three-dimensional structures in solution, while others are mainly mixtures of poorly structured disulfide isomers. The three-dimensional structures of hGrnA, hGrnC, and hGrnF contain a stable stack of two beta-hairpins in their N-terminal subdomains, but showed a more flexible C-terminal subdomain. Interestingly, of the well-structured GEMs, hGrnA demonstrated potent growth inhibition of a breast cancer cell line, while hGrnF was stimulatory. Poorly folded peptides were either weakly inhibitory or without activity. The functionally active and structurally well-characterized human hGrnA offers a unique opportunity for detailed structure-function studies of these important GEM proteins as novel members of mammalian growth factors.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Sequência de Aminoácidos , Neoplasias da Mama , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dimerização , Dissulfetos/análise , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Progranulinas , Dobramento de Proteína , Precursores de Proteínas/química , Alinhamento de Sequência
17.
J Endocrinol ; 196(1): 149-58, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18180326

RESUMO

Lys-gamma3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-gamma3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-gamma3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-gamma3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-gamma3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-gamma3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyperphosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-gamma3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.


Assuntos
Adipócitos/fisiologia , Fragmentos de Peptídeos/fisiologia , Pró-Opiomelanocortina/fisiologia , Células 3T3-L1 , Hormônio Adrenocorticotrópico/farmacologia , Animais , Proteínas de Transporte , Ésteres do Colesterol/metabolismo , Hidrólise , Lipólise/efeitos dos fármacos , Camundongos , Fragmentos de Peptídeos/farmacologia , Perilipina-1 , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Pró-Opiomelanocortina/farmacologia , Esterol Esterase/metabolismo , alfa-MSH/análogos & derivados , alfa-MSH/farmacologia
18.
J Neurochem ; 98(3): 838-50, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16893422

RESUMO

Neural apoptosis-regulated convertase-1/proprotein convertase subtilisin-kexin like-9 (NARC-1/PCSK9) is a proprotein convertase recently described to play a major role in cholesterol homeostasis through enhanced degradation of the low-density lipoprotein receptor (LDLR) and possibly in neural development. Herein, we investigated the potential involvement of this proteinase in the development of the CNS using mouse embryonal pluripotent P19 cells and the zebrafish as models. Time course quantitative RT-PCR analyses were performed following retinoic acid (RA)-induced neuroectodermal differentiation of P19 cells. Accordingly, the mRNA levels of NARC-1/PCSK9 peaked at day 2 of differentiation and fell off thereafter. In contrast, the expression of the proprotein convertases subtilisin kexin isozyme 1/site 1 protease and Furin was unaffected by RA, whereas that of PC5/6 and PC2 increased within and/or after the first 4 days of the differentiation period respectively. This pattern was not affected by the cholesterogenic transcription factor sterol regulatory element-binding protein-2, which normally up-regulates NARC-1/PCSK9 mRNA levels in liver. Furthermore, in P19 cells, RA treatment did not affect the protein level of the endogenous LDLR. This agrees with the unique expression pattern of NARC-1/PCSK9 in the rodent CNS, including the cerebellum, where the LDLR is not significantly expressed. Whole-mount in situ hybridization revealed that the pattern of expression of zebrafish NARC-1/PCSK9 is similar to that of mouse both in the CNS and periphery. Specific knockdown of zebrafish NARC-1/PCSK9 mRNA resulted in a general disorganization of cerebellar neurons and loss of hindbrain-midbrain boundaries, leading to embryonic death at approximately 96 h after fertilization. These data support a novel role for NARC-1/PCSK9 in CNS development, distinct from that in cholesterogenic organs such as liver.


Assuntos
Sistema Nervoso/enzimologia , Sistema Nervoso/crescimento & desenvolvimento , Pró-Proteína Convertase 1/fisiologia , Serina Endopeptidases/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Colesterol/biossíntese , Colesterol/genética , Humanos , Fígado/enzimologia , Camundongos , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , Pró-Proteína Convertase 1/biossíntese , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Serina Endopeptidases/biossíntese , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Peixe-Zebra
19.
BMC Genomics ; 6: 156, 2005 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-16277664

RESUMO

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Oligonucleotídeos Antissenso/genética , Proteínas de Peixe-Zebra/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Northern Blotting , Cromatografia Líquida de Alta Pressão , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/metabolismo , Dosagem de Genes , Biblioteca Gênica , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Oligonucleotídeos Antissenso/química , Filogenia , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição Gênica , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
Proteins ; 47(1): 14-24, 2002 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11870861

RESUMO

Carp granulins are members of an emerging class of proteins with a sequence motif encoding a parallel stack of two to four beta-hairpins. The carp granulin-1 protein forms a stack of four beta-hairpins, whereas its amino-terminal fragment appears to adopt a very stable stack of two beta-hairpins in solution. Here we determined a refined three-dimensional structure of this peptide fragment to examine potential conformational changes compared with the full-length protein. The structures were calculated with both a traditional method and a fast semiautomated method using ambiguous NMR distance restraints. The resulting sets of structures are very similar and show that a well-defined stack of two beta-hairpins is retained in the peptide. Conformational rearrangements compensating the loss of the carboxy-terminal subdomain of the native protein are restricted to the carboxy-terminal end of the peptide, the turn connecting the two beta-hairpins, and the Tyr(21) and Tyr(25) aromatic side chains. Further removal of the Val(1) and Ile(2) residues, which are part of the first beta-hairpin and components of two major hydrophobic clusters in the two beta-hairpin structure, results in the loss of the first beta-hairpin. The second beta-hairpin, which is closely associated with the first, retains a similar but somewhat less stable conformation. The invariable presence of the second beta-hairpin and the dependence of its stability on the first beta-hairpin suggest that the stack of two beta-hairpins may be an evolutionary conserved and autonomous folding unit. In addition, the high conformational stability makes the stack of two beta-hairpins an attractive scaffold for the development of peptide-based drug candidates.


Assuntos
Carpas , Peptídeos e Proteínas de Sinalização Intercelular , Fragmentos de Peptídeos/química , Proteínas/química , Sequência de Aminoácidos , Animais , Granulinas , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Análise Numérica Assistida por Computador , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência
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