The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Christopher J. Ward - One of the best experts on this subject based on the ideXlab platform.
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a new epitope tagged pkhd1 allele sheds light on Fibrocystin signaling
Kidney International, 2017Co-Authors: Wendy A Lea, Christopher J. WardAbstract:In this issue of Kidney International, Outeda et al. present a new epitope-tagged allele of murine Pkhd1 that allows the monitoring of functional Fibrocystin in vivo from the extreme C-terminus of the molecule. This work also shows that the removal of two-thirds of the intracellular tail of Fibrocystin does not result in cystogenesis in either the liver or kidney, with major implications for our understanding of Pkhd1 function and polycystic kidney disease in general.
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epitope tagged pkhd1 tracks the processing secretion and localization of Fibrocystin
Journal of The American Society of Nephrology, 2011Co-Authors: Jason L Bakeberg, Peter C Harris, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Marie C Hogan, Han Fang Tuan, Jan M Van Deursen, Vicente E Torres, Christopher J. WardAbstract:Mutations in the PKHD1 gene, which encodes Fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous Fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses Fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged Fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved Fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of Fibrocystin in its mature cleaved form, and scanning electron microscopy identified that Fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged Fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of Fibrocystin.
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proteolytic cleavage and nuclear translocation of Fibrocystin is regulated by intracellular ca2 and activation of protein kinase c
Journal of Biological Chemistry, 2006Co-Authors: Thomas Hiesberger, Tatyana V Masyuk, Peter C Harris, Christopher J. Ward, Eric Gourley, Andrea K Erickson, Peter Koulen, Nicholas F Larusso, Peter IgarashiAbstract:Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that Fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of Fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of Fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from Fibrocystin located in the cell membrane to the nucleus.
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Fibrocystin interacts with caml a protein involved in ca2 signaling
Biochemical and Biophysical Research Communications, 2005Co-Authors: Junko Nagano, Christopher J. Ward, Kenichiro Kitamura, Kristine M Hujer, Richard J Bram, Ulrich Hopfer, Kimio Tomita, Chunfa Huang, Tyler R MillerAbstract:Abstract The predicted structure of the autosomal recessive polycystic kidney disease protein, Fibrocystin, suggests that it may function as a receptor, but its function remains unknown. To understand its function, we searched for proteins that interact with the intracellular C-terminus of Fibrocystin using the yeast two-hybrid system. From the screening, we found calcium modulating cyclophilin ligand (CAML), a protein involved in Ca2+ signaling. Immunofluorescent analysis showed that both proteins are co-localized in the apical membrane, primary cilia, and the basal body of cells derived from the distal nephron Epitope-tagged expression constructs of both proteins were co-immunoprecipitated from COS7 cells. The intracellular C-terminus of Fibrocystin interacts with CAML, a protein with an intracellular distribution that is similar to that of PKD2. Fibrocystin may participate in regulation of intracellular Ca2+ in the distal nephron in a manner similar to PKD1 and PKD2 that are involved in autosomal dominant polycystic kidney disease.
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cellular and subcellular localization of the arpkd protein Fibrocystin is expressed on primary cilia
Human Molecular Genetics, 2003Co-Authors: Christopher J. Ward, Tatyana V Masyuk, David Yuan, Rachaneekorn Punyashthiti, Shelly Whelan, Nicholas F Larusso, Xiaofang Wang, Robert L Bacallao, Roser Torra, Vicente E TorresAbstract:Autosomal recessive polycystic kidney disease (ARPKD) is an infantile form of PKD characterized by fusiform dilation of collecting ducts and congenital hepatic fibrosis. The ARPKD gene, PKHD1, is large (approximately 470 kb; 67 exons) with a 12222 bp longest open reading frame, although multiple different splice forms may be generated. The predicted full-length ARPKD protein, Fibrocystin, is membrane bound with 4074 amino acids (447 kDa molecular weight). To characterize the pattern of Fibrocystin expression we have generated four monoclonal antibodies (mAb) to the cytoplasmic tail of the protein. Western analysis of human kidney membrane protein showed an identical pattern with each mAb; a strongly expressing large product (>450 kDa), consistent with the predicted protein size, and a weaker approximately 220 kDa band. The same large product was detected in rat and mouse kidney with lower level expression in liver. To further show that these mAbs recognize Fibrocystin, tissue from ARPKD patients was analyzed and no Fibrocystin products were detected. Immunohistochemical analysis of the developing kidney showed expression in the branching ureteric bud and collecting ducts, expression that persisted into adulthood. Biliary duct staining was found in the liver, plus staining in the pancreas and developing testis. Immunofluorescence analysis of MDCK cells showed a major site of expression in the primary cilia. Recent studies have associated the disease protein in various human and animal forms of PKD with cilia. The localization of Fibrocystin to cilia further strengthens that correlation and indicates that the primary defect in ARPKD may be linked to ciliary dysfunction.
Tatyana V Masyuk - One of the best experts on this subject based on the ideXlab platform.
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BASIC RESEARCH www.jasn.org Characterization of PKD Protein-Positive Exosome-Like
2013Co-Authors: Marie C Hogan, Anatoliy I Masyuk, Tatyana V Masyuk, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Luca Manganelli, Alexey A. Leontovich, Thomas G. Beito, Benjamin J. MaddenAbstract:Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and Fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1 � vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, Fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and Fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in Fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cell
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characterization of pkd protein positive exosome like vesicles
Journal of The American Society of Nephrology, 2009Co-Authors: Marie C Hogan, Anatoliy I Masyuk, Tatyana V Masyuk, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Luca Manganelli, Alexey A. Leontovich, Thomas G. Beito, Benjamin J. MaddenAbstract:Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and Fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, Fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene ( BBS3 ). Our proteomic analysis confirmed that cleavage of polycystin-1 and Fibrocystin occurs in vivo , in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in Fibrocystin. In vitro , these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
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cholangiocyte primary cilia in liver health and disease
Developmental Dynamics, 2008Co-Authors: Anatoliy I Masyuk, Tatyana V Masyuk, Nicholas F LarussoAbstract:The epithelial cells lining intrahepatic bile ducts (i.e., cholangiocytes), like many cell types in the body, have primary cilia extending from the apical plasma membrane into the bile ductal lumen. Cholangiocyte cilia express proteins such as polycystin-1, polycystin-2, Fibrocystin, TRPV4, P2Y12, AC6, that account for ciliary mechano-, osmo-, and chemo-sensory functions; when these processes are disturbed by mutations in genes encoding ciliary-associated proteins, liver diseases (i.e., cholangiociliopathies) result. The cholangiociliopathies include but are not limited to cystic and fibrotic liver diseases associated with mutations in genes encoding polycystin-1, polycystin-2, and Fibrocystin. In this review, we discuss the functions of cholangiocyte primary cilia, their role in the cholangiociliopathies, and potential therapeutic approaches.
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a mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation
Kidney International, 2007Co-Authors: John R Woollard, Tatyana V Masyuk, Bing Q. Huang, R Punyashtiti, S Richardson, Shelly Whelan, Donna J Lager, J Vandeursen, Vincente E Torres, Vincent H GattoneAbstract:Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein Fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional Fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1del2/del2 mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1del2/del2 mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD.
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proteolytic cleavage and nuclear translocation of Fibrocystin is regulated by intracellular ca2 and activation of protein kinase c
Journal of Biological Chemistry, 2006Co-Authors: Thomas Hiesberger, Tatyana V Masyuk, Peter C Harris, Christopher J. Ward, Eric Gourley, Andrea K Erickson, Peter Koulen, Nicholas F Larusso, Peter IgarashiAbstract:Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that Fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of Fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of Fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from Fibrocystin located in the cell membrane to the nucleus.
Marie C Hogan - One of the best experts on this subject based on the ideXlab platform.
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BASIC RESEARCH www.jasn.org Characterization of PKD Protein-Positive Exosome-Like
2013Co-Authors: Marie C Hogan, Anatoliy I Masyuk, Tatyana V Masyuk, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Luca Manganelli, Alexey A. Leontovich, Thomas G. Beito, Benjamin J. MaddenAbstract:Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and Fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1 � vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, Fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and Fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in Fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cell
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epitope tagged pkhd1 tracks the processing secretion and localization of Fibrocystin
Journal of The American Society of Nephrology, 2011Co-Authors: Jason L Bakeberg, Peter C Harris, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Marie C Hogan, Han Fang Tuan, Jan M Van Deursen, Vicente E Torres, Christopher J. WardAbstract:Mutations in the PKHD1 gene, which encodes Fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous Fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses Fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged Fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved Fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of Fibrocystin in its mature cleaved form, and scanning electron microscopy identified that Fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged Fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of Fibrocystin.
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characterization of pkd protein positive exosome like vesicles
Journal of The American Society of Nephrology, 2009Co-Authors: Marie C Hogan, Anatoliy I Masyuk, Tatyana V Masyuk, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Luca Manganelli, Alexey A. Leontovich, Thomas G. Beito, Benjamin J. MaddenAbstract:Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and Fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, Fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene ( BBS3 ). Our proteomic analysis confirmed that cleavage of polycystin-1 and Fibrocystin occurs in vivo , in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in Fibrocystin. In vitro , these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
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pkhdl1 a homolog of the autosomal recessive polycystic kidney disease gene encodes a receptor with inducible t lymphocyte expression
Human Molecular Genetics, 2003Co-Authors: Marie C Hogan, Christopher J. Ward, Vicente E Torres, Matthew D Griffin, Sandro Rossetti, Peter C HarrisAbstract:Autosomal-recessive polycystic kidney disease (ARPKD) is caused by mutation to a large gene, PKHD1, encoding a putative receptor protein, Fibrocystin. We have identified, through analysis of human genomic sequence, a PKHD1 homolog, PKHDL1, in chromosome region 8q23. The PKHDL1 transcript of 13081 bp was amplified as 16 fragments and sequenced; the sequence of the murine ortholog, Pkhdl1 (chromosome region 15B3) was also determined. PKHDL1 contains 78 exons, covers a genomic region of � 168 kb and encodes a large protein, Fibrocystin-L. Screening PKHDL1 in ARPKD patients with no PKHD1 mutations revealed several sequence variants but no clear mutations, making it unlikely that it is ARPKD-associated. Human Fibrocystin-L is predicted to be a large receptor protein (4243 aa; 466 kDa) with a signal peptide, single transmembrane domain and short cytoplasmic tail. Fibrocystin-L is homologous to Fibrocystin throughout most of the extracellular region with overall identity of 25.0% and similarity of 41.5%. Fibrocystin-L has extracellular domains similar to Fibrocystin with 14 copies of the TIG domain and two regions of significant homology to the protein TMEM2. Genomic sequence analysis identified no other full-length Fibrocystin homologs in humans, mice or other sequenced organisms. The Fugu fish has a Fibrocystin-L ortholog but no Fibrocystin, suggesting that the newly identified protein may be the ancestral form. PKHDL1 and Pkhdl1 are widely expressed at a low level in most tissues but only detected in blood-derived cell-lines. Low level expression was detected in many primary immune cell subtypes but up-regulated specifically in T lymphocytes, following activation signals, suggesting a role in cellular immunity.
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the gene mutated in autosomal recessive polycystic kidney disease encodes a large receptor like protein
Nature Genetics, 2002Co-Authors: Christopher J. Ward, Marie C Hogan, Sandro Rossetti, Xiaofang Wang, Robert L Bacallao, Denise L Walker, Tam P Sneddon, Vicky Kubly, Julie M Cunningham, Masahiko IshibashiAbstract:Autosomal recessive polycystic kidney disease (ARPKD) is characterized by dilation of collecting ducts and by biliary dysgenesis and is an important cause of renal- and liver-related morbidity and mortality. Genetic analysis of a rat with recessive polycystic kidney disease revealed an orthologous relationship between the rat locus and the ARPKD region in humans; a candidate gene was identified. A mutation was characterized in the rat and screening the 66 coding exons of the human ortholog (PKHD1) in 14 probands with ARPKD revealed 6 truncating and 12 missense mutations; 8 of the affected individuals were compound heterozygotes. The PKHD1 transcript, approximately 16 kb long, is expressed in adult and fetal kidney, liver and pancreas and is predicted to encode a large novel protein, Fibrocystin, with multiple copies of a domain shared with plexins and transcription factors. Fibrocystin may be a receptor protein that acts in collecting-duct and biliary differentiation.
Peter C Harris - One of the best experts on this subject based on the ideXlab platform.
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epitope tagged pkhd1 tracks the processing secretion and localization of Fibrocystin
Journal of The American Society of Nephrology, 2011Co-Authors: Jason L Bakeberg, Peter C Harris, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Marie C Hogan, Han Fang Tuan, Jan M Van Deursen, Vicente E Torres, Christopher J. WardAbstract:Mutations in the PKHD1 gene, which encodes Fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous Fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses Fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged Fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved Fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of Fibrocystin in its mature cleaved form, and scanning electron microscopy identified that Fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged Fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of Fibrocystin.
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proteolytic cleavage and nuclear translocation of Fibrocystin is regulated by intracellular ca2 and activation of protein kinase c
Journal of Biological Chemistry, 2006Co-Authors: Thomas Hiesberger, Tatyana V Masyuk, Peter C Harris, Christopher J. Ward, Eric Gourley, Andrea K Erickson, Peter Koulen, Nicholas F Larusso, Peter IgarashiAbstract:Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that Fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of Fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of Fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from Fibrocystin located in the cell membrane to the nucleus.
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pkhdl1 a homolog of the autosomal recessive polycystic kidney disease gene encodes a receptor with inducible t lymphocyte expression
Human Molecular Genetics, 2003Co-Authors: Marie C Hogan, Christopher J. Ward, Vicente E Torres, Matthew D Griffin, Sandro Rossetti, Peter C HarrisAbstract:Autosomal-recessive polycystic kidney disease (ARPKD) is caused by mutation to a large gene, PKHD1, encoding a putative receptor protein, Fibrocystin. We have identified, through analysis of human genomic sequence, a PKHD1 homolog, PKHDL1, in chromosome region 8q23. The PKHDL1 transcript of 13081 bp was amplified as 16 fragments and sequenced; the sequence of the murine ortholog, Pkhdl1 (chromosome region 15B3) was also determined. PKHDL1 contains 78 exons, covers a genomic region of � 168 kb and encodes a large protein, Fibrocystin-L. Screening PKHDL1 in ARPKD patients with no PKHD1 mutations revealed several sequence variants but no clear mutations, making it unlikely that it is ARPKD-associated. Human Fibrocystin-L is predicted to be a large receptor protein (4243 aa; 466 kDa) with a signal peptide, single transmembrane domain and short cytoplasmic tail. Fibrocystin-L is homologous to Fibrocystin throughout most of the extracellular region with overall identity of 25.0% and similarity of 41.5%. Fibrocystin-L has extracellular domains similar to Fibrocystin with 14 copies of the TIG domain and two regions of significant homology to the protein TMEM2. Genomic sequence analysis identified no other full-length Fibrocystin homologs in humans, mice or other sequenced organisms. The Fugu fish has a Fibrocystin-L ortholog but no Fibrocystin, suggesting that the newly identified protein may be the ancestral form. PKHDL1 and Pkhdl1 are widely expressed at a low level in most tissues but only detected in blood-derived cell-lines. Low level expression was detected in many primary immune cell subtypes but up-regulated specifically in T lymphocytes, following activation signals, suggesting a role in cellular immunity.
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Molecular Basis of Polycystic Kidney Disease: PKD1, PKD2 and PKHD1
Current opinion in nephrology and hypertension, 2002Co-Authors: Peter C HarrisAbstract:Recent developments have helped elucidate the function of the autosomal dominant polycystic kidney disease proteins, polycystin-1 and polycystin-2, and have revealed the primary defect in autosomal recessive polycystic kidney disease, by positional cloning of the gene, PKHD1. Several studies demonstrating that polycystin-2 can act as a calcium-ion-permeable cation channel, and that polycystin-1 may be involved in regulating/localizing this channel, have provided compelling evidence of the function of these proteins. A role in regulating intracellular calcium levels seems likely, with the many cellular abnormalities associated with cystogenesis due to a disruption of calcium homeostasis. Improved mutation analysis in autosomal dominant polycystic kidney disease has led to the finding of genotype/phenotype correlations which could be related to possible cleavage of polycystin-1. A major recent breakthrough has revealed the primary defect in autosomal recessive polycystic kidney disease. Genetic analysis showed that the PCK rat model is orthologous to autosomal recessive polycystic kidney disease, and allowed the human gene, PKHD1, to be precisely localized and identified. PKHD1 is a large gene, encoding a protein, Fibrocystin, of 4074 amino acids, which is predicted to have a large extracellular region, a single transmembrane domain and a short cytoplasmic tail. Fibrocystin may act as a receptor with critical roles in collecting-duct and biliary development.
Bing Q. Huang - One of the best experts on this subject based on the ideXlab platform.
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BASIC RESEARCH www.jasn.org Characterization of PKD Protein-Positive Exosome-Like
2013Co-Authors: Marie C Hogan, Anatoliy I Masyuk, Tatyana V Masyuk, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Luca Manganelli, Alexey A. Leontovich, Thomas G. Beito, Benjamin J. MaddenAbstract:Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and Fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1 � vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, Fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and Fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in Fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cell
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epitope tagged pkhd1 tracks the processing secretion and localization of Fibrocystin
Journal of The American Society of Nephrology, 2011Co-Authors: Jason L Bakeberg, Peter C Harris, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Marie C Hogan, Han Fang Tuan, Jan M Van Deursen, Vicente E Torres, Christopher J. WardAbstract:Mutations in the PKHD1 gene, which encodes Fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous Fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses Fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged Fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved Fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of Fibrocystin in its mature cleaved form, and scanning electron microscopy identified that Fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged Fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of Fibrocystin.
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characterization of pkd protein positive exosome like vesicles
Journal of The American Society of Nephrology, 2009Co-Authors: Marie C Hogan, Anatoliy I Masyuk, Tatyana V Masyuk, Bing Q. Huang, John R Woollard, Rachaneekorn Tammachote, Luca Manganelli, Alexey A. Leontovich, Thomas G. Beito, Benjamin J. MaddenAbstract:Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and Fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1 del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, Fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene ( BBS3 ). Our proteomic analysis confirmed that cleavage of polycystin-1 and Fibrocystin occurs in vivo , in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in Fibrocystin. In vitro , these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
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a mouse model of autosomal recessive polycystic kidney disease with biliary duct and proximal tubule dilatation
Kidney International, 2007Co-Authors: John R Woollard, Tatyana V Masyuk, Bing Q. Huang, R Punyashtiti, S Richardson, Shelly Whelan, Donna J Lager, J Vandeursen, Vincente E Torres, Vincent H GattoneAbstract:Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKHD1) gene encoding the protein Fibrocystin/polyductin. The aim of our study was to produce a mouse model of ARPKD in which there was no functional Fibrocystin/polyductin to study the pathophysiology of cystic and fibrocystic disease in renal and non-renal tissues. Exon 2 of the gene was deleted and replaced with a neomycin resistance cassette flanked by loxP sites, which could be subsequently removed by Cre-lox recombinase. Homozygous Pkhd1del2/del2 mice were viable, fertile and exhibited hepatic, pancreatic, and renal abnormalities. The biliary phenotype displayed progressive bile duct dilatation, resulting in grossly cystic and fibrotic livers in all animals. The primary cilia in the bile ducts of these mutant mice had structural abnormalities and were significantly shorter than those of wild-type (WT) animals. The Pkhd1del2/del2 mice often developed pancreatic cysts and some exhibited gross pancreatic enlargement. In the kidneys of affected female mice, there was tubular dilatation of the S3 segment of the proximal tubule (PT) starting at about 9 months of age, whereas male mice had normal kidneys up to 18 months of age. Inbreeding the mutation onto BALBc/J or C57BL/6J background mice resulted in females developing PT dilatation by 3 months of age. These inbred mice will be useful resources for studying the mechanisms underlying the pathogenesis of ARPKD.
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Defects in cholangiocyte Fibrocystin expression and ciliary structure in the PCK rat.
Gastroenterology, 2003Co-Authors: Tatyana V Masyuk, Anatoliy I Masyuk, Bing Q. Huang, Christopher J. Ward, David Yuan, Patrick L. Splinter, Rachaneekorn Punyashthiti, Erik L. RitmanAbstract:Abstract Background & Aims: Recent studies have showed that proteins associated with polycystic kidney disease (PKD) are expressed in cilia, linking this organelle and cyst formation in the kidney, but involvement of cilia in PKD-related biliary cystogenesis has not been shown. We investigated: (1) the expression of Fibrocystin (a product of PKHD1 , the autosomal-recessive PKD [ARPKD] gene) in cholangiocyte cilia; (2) biliary cyst formation in an orthologous rat model, PCK; and (3) the effect of Pkhd1 mutation on ciliary structure. Methods: Biliary cystogenesis was assessed by microcomputed tomography. Fibrocystin expression in cholangiocytes of isolated intrahepatic bile ducts (IBDUs) and liver cysts was analyzed by confocal and immunoelectron microscopy, and ciliary structure and length by scanning and transmission electron microscopy. Small interfering RNAs (siRNA) were used to examine the effect of Fibrocystin loss on ciliary structure. Results: The biliary tree in the PCK rat was distorted markedly, showing multiple bile duct dilatation and focal budding. In normal IBDUs, each cholangiocyte had a single cilium that expressed Fibrocystin. In contrast, cilia in the PCK rat were abnormal with bulbous extensions and diminished length, and were devoid of Fibrocystin. In cholangiocytes of normal IBDUs, specific siRNA reduced Pkhd1 messenger RNA by 80%, the length of cilia by 41%, and Fibrocystin ciliary expression to an undetectable level. Conclusions: Our results indicate that Fibrocystin is expressed in cholangiocyte cilia and that disruption of Pkhd1 by a germ line mutation in the PCK rat or by siRNA in IBDUs results in abnormalities in ciliary morphology and possibly biliary cystogenesis.