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Anton J M Roebroek

Publications and source records attributed to Anton J M Roebroek.

7 recordsLinked to original sources
PubMed2006

Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development.

Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY-->AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR-->AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL-->AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.

Alleles↗
PubMed2005

Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice.

Beta-secretase (BACE1) is the rate-limiting protease for the generation of the amyloid beta-peptide (Abeta) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1-/- lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Abeta generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Abeta generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity.

Amyloid Precursor Protein Secretases↗
PubMed2004

Limited redundancy of the proprotein convertase furin in mouse liver.

Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver. In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, alpha(5) integrin, lipoprotein receptor-related protein, vitronectin and alpha(1)-microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.

Albumins↗
PubMed2004

Low-density lipoprotein receptor-related protein interacts with MafB, a regulator of hindbrain development.

The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release. Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens. We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein. MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain. The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development.

Amino Acid Sequence↗
PubMed2003

Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule.

Megalin is a member of the LDL receptor gene family that plays an important role in forebrain development and in cellular vitamin D metabolism through endocytic uptake of vitamin D metabolites. Similar to other receptors in this gene family, megalin is believed to functionally interact with intracellular proteins through adaptors that bind to the receptor tail and regulate its endocytic and signal transducing activities. Using yeast two-hybrid screens, we identified a novel scaffold protein with tetratrico peptide repeats, the megalin-binding protein (MegBP) that associates with the receptor. The binding site of MegBP was mapped to an N-terminal region on the receptor tail harboring a proline-rich peptide element. MegBP binding did not block the endocytic activity of the receptor; however, overexpression resulted in cellular lethality. In further screens, we identified proteins that bound to MegBP and thus might be recruited to the megalin tail. MegBP-interacting partners included several transcriptional regulators such as the SKI-interacting protein (SKIP), a co-activator of the vitamin D receptor. These finding suggest a model whereby megalin directly participates in transcriptional regulation through controlled sequestration or release of transcription factors via MegBP.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences.

Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before [Biochim. Biophys. Acta 1173 (1993) 71]. The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs. These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits. To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences. Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp. Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively. After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line. Transient expression showed the expected maturation of the LRP precursor into its two subunits. Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA). Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor. This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.

Amino Acid Sequence↗