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.
神経系の発達におけるプロタンパク転換酵素麻薬の売人-1/PCSK9 の含意。 Journal of Neurochemistry. Aug, 2006 | Pubmed ID: 16893422 神経アポトーシス規制の転換酵素 1/プロタンパク転換酵素サチライシン kexin 様 9 （麻薬の売人-1/PCSK9) コレステロール恒常性低比重リポ蛋白受容体 (LDLR) の分解促進を通じて、おそらく神経の開発に大きな役割を果たすに最近説明プロタンパク転換酵素です。本明細書でこのプロティナーゼ マウス P19 多能性幹細胞と、ゼブラフィッシュをモデルにした中枢神経系の開発の潜在的な関与を検討しました。時間コース定量的 RT-PCR 解析は、レチノイン酸 (RA)、次を行った-神経外胚葉性 P19 細胞の分化誘導されます。したがって、麻薬の売人-1/PCSK9 の mRNA のレベルは分化で 2 日目をピークに、その後落ちた。対照的に、PC5/6、PC2 の内や分化期間の最初の 4 日間後それぞれ増加したに対しプロタンパクおサチライシン kexin アイソザイム 1/サイト 1 プロテアーゼと風鈴の発現は RA によって影響されなかった。このパターンは cholesterogenic 転写因子ステロール規制要素バインディング タンパク質 - 通常アップ制御 - 肝臓での麻薬の売人-1/PCSK9 mRNA のレベル 2、によって影響されなかった。さらに、P19 細胞に RA 治療タンパク質レベルの内因性の LDLR 影響を受けなかった.これは、麻薬の売人-1/PCSK9 のユニークな表現のパターンで、齧歯類、LDLR 大幅に表現されていない小脳を含む中枢神経系、同意します。全体マウント in situ ハイブリダイゼーション ゼブラフィッシュの発現パターンが麻薬の売人-1/PCSK9 マウス中枢神経系と末梢の両方のと同様であることを明らかにしました。特定のノックダウン ゼブラフィッシュ麻薬の売人-1/PCSK9 mRNA の小脳神経の一般的な無秩序と菱脳中脳の境界は、受精後約 96 h で胚の死につながるの損失をもたらした。これらのデータ CNS 開発、肝臓などの cholesterogenic の臓器にから明瞭な麻薬の売人-1/PCSK9 の新規役割をサポートします。
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.
分子クローニングと胚性発現ゼブラフィッシュ PCSK5 Co 第オーソロガス: 機能的アセスメント側線開発中。 Developmental Dynamics : an Official Publication of the American Association of Anatomists. Nov, 2010 | Pubmed ID: 20882679 Pro-protein 転換酵素サチライシン/kexin 5 (PC5 PC6 とも呼ばれます) は、セリンプロテアーゼ潜在前駆体タンパク質の成熟の機能的活性誘導体への関与のサチライシン様スーパーファミリーのメンバーです。機能的役割を調査するには、2 つ候補ゼブラフィッシュ PC5 （PCSK5.1 と PCSK5.2 として指定） お co-哺乳類で発見遺伝子 (PCSK5) のエンコーディングにまたがるオルソログ関係単一 PC5 エンコーディング cDNA 配列のクローンを作成しています。両方ポジショナルシンテニック対応 PCSK5 遺伝子を表示します。全体的に種を渡って遺伝子アーキテクチャを保存しています。PC5.1 mRNA の発現は耳胞と側線オイカワ内非常に離散的ですが、中枢神経系一緒に特定のローカリゼーションの肝臓、腸、耳胞など様々 な臓器内の PC5.2 トラン スクリプトより遍在で表されます。PC5.1 欠損ゼブラフィッシュ胚異常 neuromast 蒸着側線システム内での表示し、通常タッチ応答、側線空間意識と環境を感知で果たしている知られている感覚の役割と一貫性を欠いています。
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.