The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine.

The purpose of the study was to investigate the transepithelial transport route of Arg-Leu-Ser-Phe-Asn-Pro (RLSFNP), a milk-derived angiotensin-converting enzyme (ACE) inhibitory peptide, and to encapsulate RLSFNP in a liposome to improve its intestinal bioavailability. The transport route was investigated using transport inhibitors in a human intestinal Caco-2 cell monolayer model. Sodium azide and wortmannin significantly reduced the permeability of RLSFNP (P < 0.01), indicating that energy-dependent transcytosis is involved in the transport of RLSFNP across Caco-2 cells. The hexapeptide RLSFNP was then embedded in liposomes, and the RLSFNP liposome was characterized. Afterward, the cellular uptake and transepithelial transport ability of the RLSFNP liposome across Caco-2 cell monolayers was observed. The results demonstrated that the RLSFNP liposome was successfully prepared, having a significant sustained release and storage capability. The RLSFNP liposome can be absorbed by Caco-2 cells, with an increased intestinal absorption of RLSFNP compared to RLSFNP alone. The results showed a new way to improve RLSFNP intestinal bioavailability.

This study sought to assess the cholesterol-lowering activity of peptides obtained from milk casein hydrolyzed with neutrase. The bioactive peptides were separated using a Sephadex G-10 chromatographic column (Amersham Pharmacia Biotech, Uppsala, Sweden) after ultrafiltration using a 1-kDa molecular mass cutoff membrane. Via ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that peptides Thr- (185)(186)(187)(188)(189)(190)] reduced micellar cholesterol solubility. After Caco-2 cells were treated with LQPE, VLPVPQ, and VAPFPE, the Niemann-Pick C1-Like 1 (NPC1L1) protein levels decreased by (means ± SEM) 19.33 ± 2.47%, 52.1 ± 3.77%, and 23.09 ± 8.52%, respectively, compared with the control group. Treatment with each peptide induced significant upregulation of ATP binding cassette subfamily G member 8 antibody (ABCG8) mRNA expression by 398.1 ± 23.27%, 86.4 ± 27.07%, and 92.8 ± 8.49%. We found that VLPVPQ and LQPE significantly upregulated ATP-binding cassette transporter A1 (ABCA1) transcription by 203.9 ± 8.44% and 220.8 ± 36.42% respectively, whereas VLPVPQ significantly decreased mRNA expression of acetyl-CoA-acetyltransferase 2 (ACAT2) and microsomal triacylglycerols (MTP). The cholesterol-lowering action of milk-derived peptides may be induced by suppression of micellar cholesterol solubility and affects the expression of cholesterol absorption-related proteins and enzymes in intestinal epithelial cells. This research discovers new milk-derived peptides with decreasing cholesterol micellar solubility and provides a theoretical basis of in vitro cholesterollowering effects of peptides.

Keywords: Ape1, Atg19, autophagy, Cvt, X-ray crystallographyAbbreviations: 5 0 -IAF, 5-iodoacetamidofluorescein; Ape1, aminopeptidase I; Atg, autophagy-related; AUC, analytical ultracentrifugation; Cvt, cytoplasm-to-vacuole targeting; DDM, n-dodecyl-b-D-maltopyranoside; EM, electron microscopy; FM 4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide; FP, fluorescence polarization; GFP, green fluorescent protein; MBP, maltose binding protein; Ni-NTA, nickel-nitrilotriacetic acid; PAS, phagophore assembly site; TEV, tobacco etch virus.In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the Xray crystal structure of Ape1 at 2.5 A resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the intersubunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy.