Amyloid precursor protein (APP) is processed sequentially by the β-site APP cleaving enzyme and γ-secretase to generate amyloid β (Aβ) peptides, one of the hallmarks of Alzheimer's disease. The intracellular location of Aβ production-endosomes or the transGolgi network (TGN)-remains uncertain. We investigated the role of different postendocytic trafficking events in Aβ 40 production using an RNAi approach. Depletion of Hrs and Tsg101, acting early in the multivesicular body pathway, retained APP in early endosomes and reduced Aβ 40 production. Conversely, depletion of CHMP6 and VPS4, acting late in the pathway, rerouted endosomal APP to the TGN for enhanced APP processing. We found that VPS35 (retromer)-mediated APP recycling to the TGN was required for efficient Aβ 40 production. An interruption of the bidirectional trafficking of APP between the TGN and endosomes, particularly retromer-mediated retrieval of APP from early endosomes to the TGN, resulted in the accumulation of endocytosed APP in early endosomes with reduced APP processing. These data suggest that Aβ 40 is generated predominantly in the TGN, relying on an endocytosed pool of APP recycled from early endosomes to the TGN. endocytosis | endosomal sorting complexes required for transport pathway | vesicular traffic | protein sorting | proteolytic processing

The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.

Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRP␣, a member of the signal-regulatory proteins. We show that the variable ␤5-loop-␤6 motif confers SHP-1 substrate specificity at the P-4 and further Nterminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in proteintyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1. Protein-tyrosine phosphatases (PTPs)1 consist of a diverse family of enzymes that play crucial roles in cell growth, differentiation, and transformation (1-3). They can be broadly divided into membrane-bound, receptor-like PTPs, and cytosolic PTPs. The cytosolic PTPs contain only one catalytic domain, whereas the membrane-bound receptor-like PTPs usually contain two tandem catalytic domains. The catalytic domains of PTPs are highly conserved in their three-dimensional structures (4 -7). However, they have remarkably different substrate specificity (3, 8 -10), which is still not well understood. Previous studies using various synthetic phosphotyrosyl peptides failed to identify a shared by PTP substrate because the peptides studied were not derived from physiological substrates of PTPs. In the present study, we have addressed the structural basis for the substrate specificity of PTPs using SHP-1 and its physiological substrate SIRP␣/SHPS-1 as a model. SIRP␣ is a transmembrane protein of the signal-regulatory protein family. Its extracellular domain contains three immunoglobulin domains, and its cytoplasmic domain contains four phosphotyrosine sites (Tyr(P) 427 , Tyr(P) 452 , Tyr(P) 469 , and Tyr(P) 495 ). SHP-1 is expressed primarily in hematopoietic cells, and contains two Src homology 2 (SH2) domains, a neighboring catalytic domain, and a C-terminal tail. Its phosphatase activity is inhibited by both the SH2 domains and the C-terminal tail (11,12). SHP-1 is activated upon the binding of its tandem SH2 domains to immunoreceptor tyrosine-based inhibitory motifs. Domain-swapping studies on SHP-1 and its analogue, SHP-2, have shown that the catalytic domains of SHP-1 and SHP-2 have distinct substrate specificity (9, 10), and therefore illustrate that the dissection of the structural basis for the substrate specificity of SHP-1 is fundamental to the understanding of its physiological functions. The identification of the substrates of SHP-1 (i.e. SIRP␣, CD22, and CD72; Refs. 13-15) has made it possible for us to probe this structural basis. The results of this probe are presented below. EXPERIMENTAL PROCEDURESCrystallization and Data Collection-The C455S mutant of the SHP-1 catalytic domain (245-532) was cloned, expressed, and purified as described elsewhere (16). The phosphotyrosyl decapeptides were synthesized and purified to ...

The signal recognition particle (SRP)–dependent targeting pathway facilitates rapid, efficient delivery of the ribosome–nascent chain complex (RNC) to the protein translocation channel. We test whether the SRP receptor (SR) locates a vacant protein translocation channel by interacting with the yeast Sec61 and Ssh1 translocons. Surprisingly, the slow growth and cotranslational translocation defects caused by deletion of the transmembrane (TM) span of yeast SRβ (SRβ-ΔTM) are exaggerated when the SSH1 gene is disrupted. Disruption of the SBH2 gene, which encodes the β subunit of the Ssh1p complex, likewise causes a growth defect when combined with SRβ-ΔTM. Cotranslational translocation defects in the ssh1ΔSRβ-ΔTM mutant are explained by slow and inefficient in vivo gating of translocons by RNCs. A critical function for translocation channel β subunits in the SR–channel interaction is supported by the observation that simultaneous deletion of Sbh1p and Sbh2p causes a defect in the cotranslational targeting pathway that is similar to the translocation defect caused by deletion of either subunit of the SR.