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The presenilin complex, consisting of presenilin, nicastrin, anterior pharynx defective-1 and presenilin enhancer-2, constitutes γ-secretase, which is required for the generation of amyloid β-protein. In this article, we show that Synoviolin (also called Hrd1), which is an E3 ubiquitin ligase implicated in endoplasmic reticulum-associated degradation, is involved in the degradation of endogenous immature nicastrin, and affects amyloid β-protein generation. It was found that the level of immature nicastrin was dramatically increased in synoviolin-null cells as a result of the inhibition of degradation, but the accumulation of endogenous presenilin, anterior pharynx defective-1 and presenilin enhancer-2 was not changed. This was abolished by the transfection of exogenous Synoviolin. Moreover, nicastrin was co-immunoprecipitated with Synoviolin, strongly suggesting that nicastrin is the substrate of Synoviolin. Interestingly, amyloid β-protein generation was increased by the overexpression of Synoviolin, although the nicastrin level was decreased. Thus, Synoviolin-mediated ubiquitination is involved in the degradation of immature nicastrin, and probably regulates amyloid β-protein generation. Structured digital abstract • : Synoviolin (uniprotkb: ) physically interacts ( ) with NCT (uniprotkb: ) by anti tag coimmunoprecipitation ( ) • : Ubiquitin (uniprotkb: ) physically interacts ( ) with NCT (uniprotkb: ) by anti bait coimmunoprecipitation ( ) • : NCT (uniprotkb: ) physically interacts ( ) with Synoviolin (uniprotkb: ) by anti bait coimmunoprecipitation ( ) [ABSTRACT FROM AUTHOR]

The gene for a testicular cell adhesion protein called Tpx-1, which mediates the binding of spermatogenic cells to Sertoli cells of the rat in primary culture, was previously cloned. Here the characterization of Tpx-1 is reported. Tpx-1 messenger ribonucleic acid (mRNA) became detectable in pachytene spermatocytes and continued to be present throughout development into elongated spermatids, while the amount of Tpx-1 protein seemed to increase some time after the increment of mRNA. Tpx-1 protein was also present, although less abundantly, in spermatozoa prepared from the epididymis. Tpx-1 contains a cluster of hydrophobic amino acid residues near the amino terminus and a cysteine-rich region in the carboxyl-terminal half. Tpx-1 fused with green fluorescence protein was secreted into the medium when expressed in a cultured cell line, depending on the presence of the amino-terminal hydrophobic region. Moreover, Tpx-1 was present in the medium of testicular cell primary culture. Structure–function analysis revealed that the amino-terminal 101 amino acid residues were sufficient for cell adhesion activity, whereas the carboxyl-terminal cysteine-rich region was dispensable. In conclusion, Tpx-1 is produced and secreted from spermatogenic cells at various differentiation stages, and mediates the interaction of those cells with Sertoli cells. [ABSTRACT FROM AUTHOR]