In JoVE (1)
Other Publications (11)
- BMC Genomics
- Journal of Neurochemistry
- The Journal of Endocrinology
- Protein Science : a Publication of the Protein Society
- BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology
- BMC Neuroscience
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Journal of Molecular Neuroscience : MN
- Methods in Molecular Biology (Clifton, N.J.)
- Human Molecular Genetics
Articles by Hugh P. J. Bennett in JoVE
Other articles by Hugh P. J. Bennett on PubMed
Solution Structures of a 30-residue Amino-terminal Domain of the Carp Granulin-1 Protein and Its Amino-terminally Truncated 3-30 Subfragment: Implications for the Conformational Stability of the Stack of Two Beta-hairpins Proteins. Apr, 2002 | Pubmed ID: 11870861 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.
The Zebrafish Progranulin Gene Family and Antisense Transcripts BMC Genomics. 2005 | Pubmed ID: 16277664 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.
细菌性 Convertase 同-1/PCSK9 中枢神经系统发展中的意义。 Journal of Neurochemistry. Aug, 2006 | Pubmed ID: 16893422 神经调节细胞凋亡 convertase-1/细菌性 convertase 枯草科信类似-9 （同-1/PCSK9) 是细菌性的 convertase 最近描述了通过增强低密度脂蛋白受体 (LDLR) 降解胆固醇稳态和可能在神经发育中发挥主要作用。本文所述，我们调查了这蛋白酶作为模型使用鼠标 P19 胚胎多能干细胞和斑马鱼中枢神经系统发展中的潜在参与。时间课程定量逆转录-聚合酶链反应分析被执行之后视黄酸 (RA)-诱导 P19 细胞神经外胚层分化。因此，同-1/PCSK9 的 mRNA 水平在分化的第 2 天达到最高峰，此后掉下来了。相比之下，细菌性 convertases 枯草科信同工酶 1/站点 1 蛋白酶和弗林蛋白酶的表达是受 RA，而 PC5/6 和 PC2 分别增加内和 (或) 后第 4 天的分化期。这种模式并不受 cholesterogenic 转录因子甾醇规管元素绑定蛋白-2，其中通常向上调节肝同-1/PCSK9 mRNA 水平。此外，在 P19 细胞，RA 的待遇并不影响内源性的 LDLR 蛋白质水平。这同意同-1/PCSK9 的中枢神经系统，包括的小脑，没有明显表示 LDLR 啮齿类动物中的独特表达模式。整个装载原位杂交揭示的斑马鱼的表达模式同-1/PCSK9 是类似于鼠标在中枢神经系统和外围。斑马鱼同-1/PCSK9 mRNA 的特定 ⑷ 造成小脑神经细胞的作用一般解体和后脑中脑的界限，导致胚胎死亡约 96 h 在受精后的损失。这些数据同-1/PCSK9 支持新型的作用在中枢神经系统发育，有别于 cholesterogenic 器官如肝脏中。
Evidence of a Possible Role for Lys-gamma3-MSH in the Regulation of Adipocyte Function The Journal of Endocrinology. Jan, 2008 | Pubmed ID: 18180326 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.
Structure Dissection of Human Progranulin Identifies Well-folded Granulin/epithelin Modules with Unique Functional Activities Protein Science : a Publication of the Protein Society. Apr, 2008 | Pubmed ID: 18359860 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.
The Granulin Gene Family: from Cancer to Dementia BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Nov, 2009 | Pubmed ID: 19795409 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.
Progranulin is Expressed Within Motor Neurons and Promotes Neuronal Cell Survival BMC Neuroscience. 2009 | Pubmed ID: 19860916 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.
分子克隆和胚胎表达的斑马鱼 PCSK5 Co Orthologues： 功能评估期间侧线发展。 Developmental Dynamics : an Official Publication of the American Association of Anatomists. Nov, 2010 | Pubmed ID: 20882679 Pro-protein convertase 枯草/科信 5 (PC5，也称为 PC6） 是牵连到其功能活跃的衍生工具的潜在前体蛋白成熟的丝氨酸蛋白酶的枯草杆菌家族的成员。若要调查的职能角色，我们有克隆的 cDNA 序列编码两个候选人斑马鱼 PC5 convertases （指定为 PCSK5.1 和 PCSK5.2） co-orthologous 到单个 PC5 编码基因 (PCSK5) 在哺乳动物中找到。两者都显示人类 PCSK5 基因 syntenic 函授。整体基因结构已跨物种已保守。虽然 PC5.1 mRNA 表达是非常离散耳囊泡和侧线 neuromasts 范围内，PC5.2 成绩单更无处不在与特定的本地化，各机构包括肝、 肠、、 耳泡在中枢神经系统内表示。斑马鱼 PC5.1 缺胚胎显示异常 neuromast 侧线系统内的沉积和缺乏正常的触摸反应，与侧线在对空间的认识和感知环境的已知感官作用相一致。
Structure, Function, and Mechanism of Progranulin; the Brain and Beyond Journal of Molecular Neuroscience : MN. Nov, 2011 | Pubmed ID: 21691802 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.
Use of Zebrafish and Knockdown Technology to Define Proprotein Convertase Activity Methods in Molecular Biology (Clifton, N.J.). 2011 | Pubmed ID: 21805249 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.
Reduction of Polyglutamine Toxicity by TDP-43, FUS and Progranulin in Huntington's Disease Models Human Molecular Genetics. Feb, 2013 | Pubmed ID: 23172908 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.