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Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease.

Kajiwara Y, Franciosi S, Takahashi N, Krug L, Schmeidler J, Taddei K, Haroutunian V, Fried U, Ehrlich M, Martins RN, Gandy S, Buxbaum JD - Mol Neurodegener (2010)

Bottom Line: Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression.These results implicate NYGGF4 as a novel and specific interactor of LRP1.The results support further studies on the functional relationship between NYGGF4 and LRP1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Neuropsychiatry, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA. joseph.buxbaum@mssm.edu.

ABSTRACT

Background: The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.

Results: We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP), which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b). Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression.

Conclusions: These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered early in disease. Genetic and functional studies have implicated both LRP1 and NYGGF4 in obesity and cardiovascular disease and the physical association of these proteins may reflect a common mechanism. This is particularly interesting in light of the dual role of ApoE in both cardiovascular risk and AD. The results support further studies on the functional relationship between NYGGF4 and LRP1.

No MeSH data available.


Related in: MedlinePlus

Mammalian two-hybrid assays with the LRP1 or APP cytoplasmic domains. H4 human neuroglioma cells were transfected with the indicated constructs, and luciferase activity measured. A. The LRP1 cytoplasmic domain was cloned into the pBIND vector while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into the pACT vector. Strong interactions between LRP1 and JIP-1b, EB-1 or Nyggf4 were observed, confirming results with yeast two hybrid studies. B. The APP cytoplasmic domain (known as APP intracellular domain/AICD) was cloned into pBIND, while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into pACT. Strong interactions between JIP-1b or EB-1 with the APP intracellular domain were observed but no interaction was observed with Nyggf4 and the APP intracellular domain. ***, P < 0.0001.
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Figure 2: Mammalian two-hybrid assays with the LRP1 or APP cytoplasmic domains. H4 human neuroglioma cells were transfected with the indicated constructs, and luciferase activity measured. A. The LRP1 cytoplasmic domain was cloned into the pBIND vector while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into the pACT vector. Strong interactions between LRP1 and JIP-1b, EB-1 or Nyggf4 were observed, confirming results with yeast two hybrid studies. B. The APP cytoplasmic domain (known as APP intracellular domain/AICD) was cloned into pBIND, while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into pACT. Strong interactions between JIP-1b or EB-1 with the APP intracellular domain were observed but no interaction was observed with Nyggf4 and the APP intracellular domain. ***, P < 0.0001.

Mentions: The interaction between LRP1 and Nyggf4 was confirmed by four additional methods. We first used a mammalian two-hybrid system to confirm that LRP1 and Nyggf4 interact in mammalian cells (Fig. 2A). Nyggf4 showed significant interaction with the LRP1 cytoplasmic domain in this assay. For comparison, we included additional potential LRP1 interactors, identified in the screen with the LRP1-C2 bait. From these latter studies, we confirmed that both EB-1 and JIP1b were LRP1 interactors in this system (Fig. 2A).


Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease.

Kajiwara Y, Franciosi S, Takahashi N, Krug L, Schmeidler J, Taddei K, Haroutunian V, Fried U, Ehrlich M, Martins RN, Gandy S, Buxbaum JD - Mol Neurodegener (2010)

Mammalian two-hybrid assays with the LRP1 or APP cytoplasmic domains. H4 human neuroglioma cells were transfected with the indicated constructs, and luciferase activity measured. A. The LRP1 cytoplasmic domain was cloned into the pBIND vector while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into the pACT vector. Strong interactions between LRP1 and JIP-1b, EB-1 or Nyggf4 were observed, confirming results with yeast two hybrid studies. B. The APP cytoplasmic domain (known as APP intracellular domain/AICD) was cloned into pBIND, while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into pACT. Strong interactions between JIP-1b or EB-1 with the APP intracellular domain were observed but no interaction was observed with Nyggf4 and the APP intracellular domain. ***, P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823744&req=5

Figure 2: Mammalian two-hybrid assays with the LRP1 or APP cytoplasmic domains. H4 human neuroglioma cells were transfected with the indicated constructs, and luciferase activity measured. A. The LRP1 cytoplasmic domain was cloned into the pBIND vector while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into the pACT vector. Strong interactions between LRP1 and JIP-1b, EB-1 or Nyggf4 were observed, confirming results with yeast two hybrid studies. B. The APP cytoplasmic domain (known as APP intracellular domain/AICD) was cloned into pBIND, while the interactors JIP-1b, EB-1 and Nyggf4 were cloned into pACT. Strong interactions between JIP-1b or EB-1 with the APP intracellular domain were observed but no interaction was observed with Nyggf4 and the APP intracellular domain. ***, P < 0.0001.
Mentions: The interaction between LRP1 and Nyggf4 was confirmed by four additional methods. We first used a mammalian two-hybrid system to confirm that LRP1 and Nyggf4 interact in mammalian cells (Fig. 2A). Nyggf4 showed significant interaction with the LRP1 cytoplasmic domain in this assay. For comparison, we included additional potential LRP1 interactors, identified in the screen with the LRP1-C2 bait. From these latter studies, we confirmed that both EB-1 and JIP1b were LRP1 interactors in this system (Fig. 2A).

Bottom Line: Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression.These results implicate NYGGF4 as a novel and specific interactor of LRP1.The results support further studies on the functional relationship between NYGGF4 and LRP1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Neuropsychiatry, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA. joseph.buxbaum@mssm.edu.

ABSTRACT

Background: The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.

Results: We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP), which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b). Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression.

Conclusions: These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered early in disease. Genetic and functional studies have implicated both LRP1 and NYGGF4 in obesity and cardiovascular disease and the physical association of these proteins may reflect a common mechanism. This is particularly interesting in light of the dual role of ApoE in both cardiovascular risk and AD. The results support further studies on the functional relationship between NYGGF4 and LRP1.

No MeSH data available.


Related in: MedlinePlus