Publications by authors named "Petr Kasparek"

21 Publications

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Response to correspondence on "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation".

Authors:
Channabasavaiah B Gurumurthy Aidan R O'Brien Rolen M Quadros John Adams Pilar Alcaide Shinya Ayabe Johnathan Ballard Surinder K Batra Marie-Claude Beauchamp Kathleen A Becker Guillaume Bernas David Brough Francisco Carrillo-Salinas Wesley Chan Hanying Chen Ruby Dawson Victoria DeMambro Jinke D'Hont Katharine Dibb James D Eudy Lin Gan Jing Gao Amy Gonzales Anyonya Guntur Huiping Guo Donald W Harms Anne Harrington Kathryn E Hentges Neil Humphreys Shiho Imai Hideshi Ishii Mizuho Iwama Eric Jonasch Michelle Karolak Bernard Keavney Nay-Chi Khin Masamitsu Konno Yuko Kotani Yayoi Kunihiro Imayavaramban Lakshmanan Catherine Larochelle Catherine B Lawrence Lin Li Volkhard Lindner Xian-De Liu Gloria Lopez-Castejon Andrew Loudon Jenna Lowe Loydie Jerome-Majeweska Taiji Matsusaka Hiromi Miura Yoshiki Miyasaka Benjamin Morpurgo Katherine Motyl Yo-Ichi Nabeshima Koji Nakade Toshiaki Nakashiba Kenichi Nakashima Yuichi Obata Sanae Ogiwara Mariette Ouellet Leif Oxburgh Sandra Piltz Ilka Pinz Moorthy P Ponnusamy David Ray Ronald J Redder Clifford J Rosen Nikki Ross Mark T Ruhe Larisa Ryzhova Ane M Salvador Sabrina Shameen Alam Radislav Sedlacek Karan Sharma Chad Smith Katrien Staes Lora Starrs Fumihiro Sugiyama Satoru Takahashi Tomohiro Tanaka Andrew Trafford Yoshihiro Uno Leen Vanhoutte Frederique Vanrockeghem Brandon J Willis Christian S Wright Yuko Yamauchi Xin Yi Kazuto Yoshimi Xuesong Zhang Yu Zhang Masato Ohtsuka Satyabrata Das Daniel J Garry Tino Hochepied Paul Thomas Jan Parker-Thornburg Antony D Adamson Atsushi Yoshiki Jean-Francois Schmouth Andrei Golovko William R Thompson K C Kent Lloyd Joshua A Wood Mitra Cowan Tomoji Mashimo Seiya Mizuno Hao Zhu Petr Kasparek Lucy Liaw Joseph M Miano Gaetan Burgio

Genome Biol 2021 Apr 7;22(1):99. Epub 2021 Apr 7.

Department of Immunology and Infectious Disease, the John Curtin School of Medical Research, The Australian National University, Canberra, Australia.

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http://dx.doi.org/10.1186/s13059-021-02320-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025318PMC
April 2021

Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance.

EMBO Rep 2021 Feb 11;22(2):e50785. Epub 2021 Jan 11.

Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS-induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.
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http://dx.doi.org/10.15252/embr.202050785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857422PMC
February 2021

Intronic Determinants Coordinate Charme lncRNA Nuclear Activity through the Interaction with MATR3 and PTBP1.

Cell Rep 2020 Dec;33(12):108548

Department of Biology and Biotechnology "Charles Darwin," Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Electronic address:

Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.
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http://dx.doi.org/10.1016/j.celrep.2020.108548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773549PMC
December 2020

The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner.

Int J Mol Sci 2020 Aug 19;21(17). Epub 2020 Aug 19.

Institute of Molecular Pharmacology, Medical Faculty, RWTH Aachen University, 52074 Aachen, Germany.

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria ( and , respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of . The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics.
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http://dx.doi.org/10.3390/ijms21175978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503280PMC
August 2020

Efficient allele conversion in mouse zygotes and primary cells based on electroporation of Cre protein.

Methods 2020 Jul 24. Epub 2020 Jul 24.

Czech Centre of Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic; Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic.

Cre-loxP recombination system is a powerful tool for genome engineering. One of its applications is found in genetic mouse models that often require to induce Cre recombination in preimplantation embryos. Here, we describe a technically simple, affordable and highly efficient protocol for Cre protein delivery into mouse zygotes by electroporation. We show that electroporation based delivery of Cre has no negative impact on embryo survival and the method can be easily combined with in vitro fertilization resulting in a significantly faster generation of desired models. Lastly, we demonstrate that Cre protein electroporation is suitable for allelic conversion in primary cells derived from conditional mouse models.
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http://dx.doi.org/10.1016/j.ymeth.2020.07.005DOI Listing
July 2020

CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review.

Cells 2020 04 16;9(4). Epub 2020 Apr 16.

Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the CAS, v.v.i, 252 50 Vestec, Czech Republic.

Imprinting diseases (IDs) are rare congenital disorders caused by aberrant dosages of imprinted genes. Rare IDs are comprised by a group of several distinct disorders that share a great deal of homology in terms of genetic etiologies and symptoms. Disruption of genetic or epigenetic mechanisms can cause issues with regulating the expression of imprinted genes, thus leading to disease. Genetic mutations affect the imprinted genes, duplications, deletions, and uniparental disomy (UPD) are reoccurring phenomena causing imprinting diseases. Epigenetic alterations on methylation marks in imprinting control centers (ICRs) also alters the expression patterns and the majority of patients with rare IDs carries intact but either silenced or overexpressed imprinted genes. Canonical CRISPR/Cas9 editing relying on double-stranded DNA break repair has little to offer in terms of therapeutics for rare IDs. Instead CRISPR/Cas9 can be used in a more sophisticated way by targeting the epigenome. Catalytically dead Cas9 (dCas9) tethered with effector enzymes such as DNA de- and methyltransferases and histone code editors in addition to systems such as CRISPRa and CRISPRi have been shown to have high epigenome editing efficiency in eukaryotic cells. This new era of CRISPR epigenome editors could arguably be a game-changer for curing and treating rare IDs by refined activation and silencing of disturbed imprinted gene expression. This review describes major CRISPR-based epigenome editors and points out their potential use in research and therapy of rare imprinting diseases.
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http://dx.doi.org/10.3390/cells9040993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226972PMC
April 2020

CRMP2 mediates Sema3F-dependent axon pruning and dendritic spine remodeling.

EMBO Rep 2020 03 9;21(3):e48512. Epub 2020 Jan 9.

Department of Molecular Neurobiology, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.

Regulation of axon guidance and pruning of inappropriate synapses by class 3 semaphorins are key to the development of neural circuits. Collapsin response mediator protein 2 (CRMP2) has been shown to regulate axon guidance by mediating semaphorin 3A (Sema3A) signaling; however, nothing is known about its role in synapse pruning. Here, using newly generated crmp2 mice we demonstrate that CRMP2 has a moderate effect on Sema3A-dependent axon guidance in vivo, and its deficiency leads to a mild defect in axon guidance in peripheral nerves and the corpus callosum. Surprisingly, crmp2 mice display prominent defects in stereotyped axon pruning in hippocampus and visual cortex and altered dendritic spine remodeling, which is consistent with impaired Sema3F signaling and with models of autism spectrum disorder (ASD). We demonstrate that CRMP2 mediates Sema3F signaling in primary neurons and that crmp2 mice display ASD-related social behavior changes in the early postnatal period as well as in adults. Together, we demonstrate that CRMP2 mediates Sema3F-dependent synapse pruning and its dysfunction shares histological and behavioral features of ASD.
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http://dx.doi.org/10.15252/embr.201948512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054682PMC
March 2020

Truncated PPM1D impairs stem cell response to genotoxic stress and promotes growth of APC-deficient tumors in the mouse colon.

Cell Death Dis 2019 10 28;10(11):818. Epub 2019 Oct 28.

Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1D allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1D resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apc mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcPpm1d mice were less sensitive to 5-fluorouracil when compared to ApcPpm1dand the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.
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http://dx.doi.org/10.1038/s41419-019-2057-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817818PMC
October 2019

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

Authors:
Channabasavaiah B Gurumurthy Aidan R O'Brien Rolen M Quadros John Adams Pilar Alcaide Shinya Ayabe Johnathan Ballard Surinder K Batra Marie-Claude Beauchamp Kathleen A Becker Guillaume Bernas David Brough Francisco Carrillo-Salinas Wesley Chan Hanying Chen Ruby Dawson Victoria DeMambro Jinke D'Hont Katharine M Dibb James D Eudy Lin Gan Jing Gao Amy Gonzales Anyonya R Guntur Huiping Guo Donald W Harms Anne Harrington Kathryn E Hentges Neil Humphreys Shiho Imai Hideshi Ishii Mizuho Iwama Eric Jonasch Michelle Karolak Bernard Keavney Nay-Chi Khin Masamitsu Konno Yuko Kotani Yayoi Kunihiro Imayavaramban Lakshmanan Catherine Larochelle Catherine B Lawrence Lin Li Volkhard Lindner Xian-De Liu Gloria Lopez-Castejon Andrew Loudon Jenna Lowe Loydie A Jerome-Majewska Taiji Matsusaka Hiromi Miura Yoshiki Miyasaka Benjamin Morpurgo Katherine Motyl Yo-Ichi Nabeshima Koji Nakade Toshiaki Nakashiba Kenichi Nakashima Yuichi Obata Sanae Ogiwara Mariette Ouellet Leif Oxburgh Sandra Piltz Ilka Pinz Moorthy P Ponnusamy David Ray Ronald J Redder Clifford J Rosen Nikki Ross Mark T Ruhe Larisa Ryzhova Ane M Salvador Sabrina Shameen Alam Radislav Sedlacek Karan Sharma Chad Smith Katrien Staes Lora Starrs Fumihiro Sugiyama Satoru Takahashi Tomohiro Tanaka Andrew W Trafford Yoshihiro Uno Leen Vanhoutte Frederique Vanrockeghem Brandon J Willis Christian S Wright Yuko Yamauchi Xin Yi Kazuto Yoshimi Xuesong Zhang Yu Zhang Masato Ohtsuka Satyabrata Das Daniel J Garry Tino Hochepied Paul Thomas Jan Parker-Thornburg Antony D Adamson Atsushi Yoshiki Jean-Francois Schmouth Andrei Golovko William R Thompson K C Kent Lloyd Joshua A Wood Mitra Cowan Tomoji Mashimo Seiya Mizuno Hao Zhu Petr Kasparek Lucy Liaw Joseph M Miano Gaetan Burgio

Genome Biol 2019 08 26;20(1):171. Epub 2019 Aug 26.

Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.

Background: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).

Results: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.

Conclusion: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.
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http://dx.doi.org/10.1186/s13059-019-1776-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709553PMC
August 2019

A novel PSMA/GCPII-deficient mouse model shows enlarged seminal vesicles upon aging.

Prostate 2019 02 5;79(2):126-139. Epub 2018 Sep 5.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague 6, Czech Republic.

Background: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is an important diagnostic and therapeutic target in prostate cancer. PSMA/GCPII is also expressed in many healthy tissues, but its function has only been established in the brain and small intestine. Several research groups have attempted to produce PSMA/GCPII-deficient mice to study the physiological role of PSMA/GCPII in detail. The outcomes of these studies differ dramatically, ranging from embryonic lethality to production of viable PSMA/GCPII-deficient mice without any obvious phenotype.

Methods: We produced PSMA/GCPII-deficient mice (hereafter also referred as Folh1 mice) by TALEN-mediated mutagenesis on a C57BL/6NCrl background. Using Western blot and an enzyme activity assay, we confirmed the absence of PSMA/GCPII in our Folh1 mice. We performed anatomical and histopathological examination of selected tissues with a focus on urogenital system. We also examined the PSMA/GCPII expression profile within the mouse urogenital system using an enzyme activity assay and confirmed the presence of PSMA/GCPII in selected tissues by immunohistochemistry.

Results: Our Folh1 mice are viable, breed normally, and do not show any obvious phenotype. Nevertheless, aged Folh1 mice of 69-72 weeks exhibit seminal vesicle dilation, which is caused by accumulation of luminal fluid. This phenotype was also observed in Folh1 mice; the overall difference between our three cohorts (Folh1 , Folh1 , and Folh1 ) was highly significant (P < 0.002). Of all studied tissues of the mouse urogenital system, only the epididymis appeared to have a physiologically relevant level of PSMA/GCPII expression. Additional experiments demonstrated that PSMA/GCPII is also present in the human epididymis.

Conclusions: In this study, we provide the first evidence characterizing the reproductive tissue phenotype of PSMA/GCPII-deficient mice. These findings will help lay the groundwork for future studies to reveal PSMA/GCPII function in human reproduction.
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http://dx.doi.org/10.1002/pros.23717DOI Listing
February 2019

Profiling system for skin kallikrein proteolysis applied in gene-deficient mouse models.

Biol Chem 2018 09;399(9):1085-1089

Laboratory of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, Prumyslova 595, 252 50 Vestec, Czech Republic.

Kallikrein-related proteases (KLKs) play a critical role in epidermis physiology and have been implicated in skin pathologies such as Netherton syndrome. The contribution of individual KLKs to skin proteolysis is poorly understood. Monitoring of their activities in skin proteome is hampered by overlapping substrate specificities, and there is a need for novel assays. Here, we present a platform of selective and sensitive fluorogenic substrates and inhibitors for profiling KLK5, KLK7 and KLK14. These chemical tools were evaluated using recombinant KLKs and tissue from a unique set of mice deficient in eight combinations of KLKs and their natural regulator LEKTI.
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http://dx.doi.org/10.1515/hsz-2018-0116DOI Listing
September 2018

Kallikrein-related peptidase 5 and seasonal influenza viruses, limitations of the experimental models for activating proteases.

Biol Chem 2018 09;399(9):1053-1064

INSERM U1100, Centre d'Etude des Pathologies Respiratoires, Faculté de Médecine, F-37032 Tours, France.

Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.
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http://dx.doi.org/10.1515/hsz-2017-0340DOI Listing
September 2018

In vivo regulation of the A disintegrin and metalloproteinase 10 (ADAM10) by the tetraspanin 15.

Cell Mol Life Sci 2018 09 8;75(17):3251-3267. Epub 2018 Mar 8.

Institute of Biochemistry, Christian Albrechts University Kiel, Olshausenstrasse 40, 24118, Kiel, Germany.

A disintegrin and metalloproteinase 10 (ADAM10) plays a major role in the ectodomain shedding of important surface molecules with physiological and pathological relevance including the amyloid precursor protein (APP), the cellular prion protein, and different cadherins. Despite its therapeutic potential, there is still a considerable lack of knowledge how this protease is regulated. We have previously identified tetraspanin15 (Tspan15) as a member of the TspanC8 family to specifically associate with ADAM10. Cell-based overexpression experiments revealed that this binding affected the maturation process and surface expression of the protease. Our current study shows that Tspan15 is abundantly expressed in mouse brain, where it specifically interacts with endogenous ADAM10. Tspan15 knockout mice did not reveal an overt phenotype but showed a pronounced decrease of the active and mature form of ADAM10, an effect which augmented with aging. The decreased expression of active ADAM10 correlated with an age-dependent reduced shedding of neuronal (N)-cadherin and the cellular prion protein. APP α-secretase cleavage and the expression of Notch-dependent genes were not affected by the loss of Tspan15, which is consistent with the hypothesis that different TspanC8s cause ADAM10 to preferentially cleave particular substrates. Analyzing spine morphology revealed no obvious differences between Tspan15 knockout and wild-type mice. However, Tspan15 expression was elevated in brains of an Alzheimer's disease mouse model and of patients, suggesting that upregulation of Tspan15 expression reflects a cellular response in a disease state. In conclusion, our data show that Tspan15 and most likely also other members of the TspanC8 family are central modulators of ADAM10-mediated ectodomain shedding in vivo.
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http://dx.doi.org/10.1007/s00018-018-2791-2DOI Listing
September 2018

Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif.

Proc Natl Acad Sci U S A 2017 02 14;114(9):E1641-E1650. Epub 2017 Feb 14.

Institute of Microbiology of the Czech Academy of Sciences, v.v.i., 142 20 Prague 4, Czech Republic;

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.
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http://dx.doi.org/10.1073/pnas.1615334114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338493PMC
February 2017

KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype.

PLoS Genet 2017 01 17;13(1):e1006566. Epub 2017 Jan 17.

Laboratory of Transgenic Models of Diseases, Division BIOCEV, Institute of Molecular Genetics of the Czech Academy of Sciences, v.v.i., Vestec, Czech Republic.

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.
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http://dx.doi.org/10.1371/journal.pgen.1006566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283769PMC
January 2017

Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

Biochim Biophys Acta Mol Cell Res 2017 Jan 3;1864(1):217-230. Epub 2016 Nov 3.

Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany. Electronic address:

Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.
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http://dx.doi.org/10.1016/j.bbamcr.2016.11.003DOI Listing
January 2017

A viable mouse model for Netherton syndrome based on mosaic inactivation of the Spink5 gene.

Biol Chem 2016 12;397(12):1287-1292

Netherton syndrome (NS) is caused by mutations in the SPINK5 gene. Several Spink5-deficient mouse models were generated to understand the mechanisms of NS in vivo. However, Spink5-deficiency in mice is associated with postnatal lethality that hampers further analysis. Here we present a viable mouse model for NS generated by mosaic inactivation of the Spink5 gene. We propose that these mice are a valuable experimental tool to study NS, especially for long-term studies evaluating potential therapeutic compounds. Furthermore, we show that mosaic inactivation of a gene using TALENs or CRISPR/Cas9 systems can be used to study lethal phenotypes in adult mice.
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http://dx.doi.org/10.1515/hsz-2016-0194DOI Listing
December 2016

Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases.

FEBS Lett 2014 Nov 18;588(21):3982-8. Epub 2014 Sep 18.

Institute of Molecular Genetics of the AS CR, v.v.i., Videnska 1083, 142 20, Czech Republic. Electronic address:

Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.
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http://dx.doi.org/10.1016/j.febslet.2014.09.014DOI Listing
November 2014

Transgenic mouse model expressing tdTomato under involucrin promoter as a tool for analysis of epidermal differentiation and wound healing.

Transgenic Res 2012 Jun 22;21(3):683-9. Epub 2011 Oct 22.

Department of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.

The epidermis is a stratified tissue composed of different keratinocyte layers that create a barrier protecting the body from external influences, pathogens, and dehydration. The barrier function is mainly achieved by its outermost layer, the stratum corneum. To create a mouse model to study pathophysiological processes in the outermost layers of the epidermis in vivo and in vitro we prepared a construct containing red fluorescent td-Tomato reporter sequence under the control of involucrin promoter and its first intron. Transgenic mice were generated by pronuclear injection and the expression and regulation of the transgene was determined by in vivo imaging and fluorescent microscopy. The promoter targeted the transgene efficiently and specifically into the outermost epidermal layers although weak expression was also found in epithelia of tongue and bladder. The regulation of expression in the epidermis, i.e. fluorescence intensity of the reporter, could be easily followed during wound healing and dermatitis. Thus, these transgenic mice carrying the tdTomato reporter could be used as a valuable tool to study impact of various genes dysregulating the epidermal barrier and to follow effects of therapeutic agents for treatment of skin diseases in vivo.
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http://dx.doi.org/10.1007/s11248-011-9567-xDOI Listing
June 2012

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812.

Proteomics 2007 Jan;7(1):64-72

Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Kotlárská 2, Brno, Czech Republic.

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.
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http://dx.doi.org/10.1002/pmic.200600280DOI Listing
January 2007