The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus

Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.

Download Full-text

The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes

International Journal of Molecular Sciences ◽

10.3390/ijms22189854 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9854

Author(s):

Christine Loo ◽

Michael Pearen ◽

Grant Ramm

Keyword(s):

Human Development ◽

Sonic Hedgehog ◽

Mammalian Cells ◽

Animal Studies ◽

Primary Cilia ◽

Genetic Mutation ◽

Wide Variability ◽

Using Data ◽

Differentiation And Proliferation

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.

Download Full-text

Molecular Pathways and Therapies in Autosomal-Dominant Polycystic Kidney Disease

Physiology ◽

10.1152/physiol.00032.2014 ◽

2015 ◽

Vol 30 (3) ◽

pp. 195-207 ◽

Cited By ~ 22

Author(s):

Takamitsu Saigusa ◽

P. Darwin Bell

Keyword(s):

Kidney Disease ◽

Polycystic Kidney Disease ◽

Primary Cilia ◽

Autosomal Dominant ◽

Molecular Pathways ◽

Polycystic Kidney ◽

Autosomal Dominant Polycystic Kidney ◽

Multiple Cysts ◽

Review Current

Autosomal-dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can eventually lead to kidney failure. Studies investigating the role of primary cilia and polycystins have significantly advanced our understanding of the pathogenesis of PKD. This review will present clinical and basic aspects of ADPKD, review current concepts of PKD pathogenesis, evaluate potential therapeutic targets, and highlight challenges for future clinical studies.

Download Full-text

Functional genetic encoding of sulfotyrosine in mammalian cells

Nature Communications ◽

10.1038/s41467-020-18629-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xinyuan He ◽

Yan Chen ◽

Daisy Guiza Beltran ◽

Maia Kelly ◽

Bin Ma ◽

...

Keyword(s):

Mammalian Cells ◽

Biochemical Characterization ◽

Trna Synthetase ◽

Functional Verification ◽

Cellular Processes ◽

Genetic Encoding ◽

Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Download Full-text

Cytoplasmic Dynein Nucleates Microtubules to Organize Them into Radial Arrays In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e03-10-0770 ◽

2004 ◽

Vol 15 (6) ◽

pp. 2742-2749 ◽

Cited By ~ 34

Author(s):

Viacheslav Malikov ◽

Anna Kashina ◽

Vladimir Rodionov

Keyword(s):

Cytoplasmic Dynein ◽

Exact Function ◽

Necessary And Sufficient ◽

Stimulation Of ◽

In Cells

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.

Download Full-text

Physiological significance of volume-regulatory transporters

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.5.c995 ◽

1999 ◽

Vol 276 (5) ◽

pp. C995-C1011 ◽

Cited By ~ 164

Author(s):

W. Charles O’Neill

Keyword(s):

Cell Volume ◽

Volume Regulation ◽

Mammalian Cells ◽

Ion Transporters ◽

Subsequent Growth ◽

The Past ◽

Shrinkage And Swelling ◽

Acute Changes

Research over the past 25 years has identified specific ion transporters and channels that are activated by acute changes in cell volume and that serve to restore steady-state volume. The mechanism by which cells sense changes in cell volume and activate the appropriate transporters remains a mystery, but recent studies are providing important clues. A curious aspect of volume regulation in mammalian cells is that it is often absent or incomplete in anisosmotic media, whereas complete volume regulation is observed with isosmotic shrinkage and swelling. The basis for this may lie in an important role of intracellular Cl− in controlling volume-regulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity but rather changes in the cellular content of osmotically active molecules. Volume-regulatory transporters are also closely linked to cell growth and metabolism, producing requisite changes in cell volume that may also signal subsequent growth and metabolic events. Thus, despite the relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology.

Download Full-text

The N-Terminus ofDictyosteliumScar Interacts with Abi and HSPC300 and Is Essential for Proper Regulation and Function

Molecular Biology of the Cell ◽

10.1091/mbc.e06-06-0518 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1609-1620 ◽

Cited By ~ 12

Author(s):

Diana Caracino ◽

Cheryl Jones ◽

Mark Compton ◽

Charles L. Saxe

Keyword(s):

Actin Polymerization ◽

Terminal Region ◽

Wild Type ◽

Large Molecular Weight ◽

Weight Protein ◽

And Function ◽

Necessary And Sufficient

Scar/WAVE proteins, members of the conserved Wiskott-Aldrich syndrome (WAS) family, promote actin polymerization by activating the Arp2/3 complex. A number of proteins, including a complex containing Nap1, PIR121, Abi1/2, and HSPC300, interact with Scar/WAVE, though the role of this complex in regulating Scar function remains unclear. Here we identify a short N-terminal region of Dictyostelium Scar that is necessary and sufficient for interaction with HSPC300 and Abi in vitro. Cells expressing Scar lacking this N-terminal region show abnormalities in F-actin distribution, cell morphology, movement, and cytokinesis. This is true even in the presence of wild-type Scar. The data suggest that the first 96 amino acids of Scar are necessary for participation in a large-molecular-weight protein complex, and that this Scar-containing complex is responsible for the proper localization and regulation of Scar. The presence of mis-regulated or unregulated Scar has significant deleterious effects on cells and may explain the need to keep Scar activity tightly controlled in vivo either by assembly in a complex or by rapid degradation.

Download Full-text

A Role for Fis1 in Both Mitochondrial and Peroxisomal Fission in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0159 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5077-5086 ◽

Cited By ~ 198

Author(s):

Annett Koch ◽

Yisang Yoon ◽

Nina A. Bonekamp ◽

Mark A. McNiven ◽

Michael Schrader

Keyword(s):

Mammalian Cells ◽

Small Interfering Rna ◽

Transmembrane Domain ◽

Mitochondrial Fission ◽

Ectopic Expression ◽

Mammalian Homologue ◽

Necessary And Sufficient ◽

Interfering Rna ◽

Peroxisome Division

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

Download Full-text

Chromatin structure and function: the heretical path to an RNA transcription factor

Biochemistry and Cell Biology ◽

10.1139/o96-067 ◽

1996 ◽

Vol 74 (5) ◽

pp. 623-632 ◽

Cited By ~ 5

Author(s):

Margarida O. Krause

Keyword(s):

Chromatin Structure ◽

Mammalian Cells ◽

T Antigen ◽

Temperature Sensitive ◽

Mobility Shift ◽

Cell Extracts ◽

Myc Gene ◽

And Function

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.

Download Full-text

Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence

Molecular and Cellular Biology ◽

10.1128/mcb.00720-06 ◽

2007 ◽

Vol 27 (10) ◽

pp. 3651-3666 ◽

Cited By ~ 36

Author(s):

Justina D. McEvoy ◽

Uta Kossatz ◽

Nisar Malek ◽

Jeffrey D. Singer

Keyword(s):

Mammalian Cells ◽

Gene Deletion ◽

Cyclin E ◽

E3 Ligase ◽

Degradation Pathway ◽

Conditional Knockout ◽

Protein Levels ◽

Primary Fibroblasts

ABSTRACT Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells.

Download Full-text