ATP13A1 prevents ERAD of folding-competent mislocalized and misoriented proteins.

The biosynthesis of thousands of proteins requires targeting a signal sequence or transmembrane segment (TM) to the endoplasmic reticulum (ER). These hydrophobic ɑ helices must localize to the appropriate cellular membrane and integrate in the correct topology to maintain a high-fidelity proteome. Here, we show that the P5A-ATPase ATP13A1 prevents the accumulation of mislocalized and misoriented proteins, which are eliminated by different ER-associated degradation (ERAD) pathways in mammalian cells. Without ATP13A1, mitochondrial tail-anchored proteins mislocalize to the ER through the ER membrane protein complex and are cleaved by signal peptide peptidase for ERAD. ATP13A1 also facilitates the topogenesis of a subset of proteins with an N-terminal TM or signal sequence that should insert into the ER membrane with a cytosolic N terminus. Without ATP13A1, such proteins accumulate in the wrong orientation and are targeted for ERAD by distinct ubiquitin ligases. Thus, ATP13A1 prevents ERAD of diverse proteins capable of proper folding.

Annotation of the Complete Genome Sequences of Bacteriophages Sara and Birdfeeder.

Sara is a siphovirus with a linear 17,362bp genome containing 25 genes. Birdfeeder is a podovirus with a circularly permuted 53,897bp genome containing 52 genes. Sara and Birdfeeder were isolated from environmental samples in Plattsburgh, NY, USA and Forest Hill, MD, USA, respectively, using Microbacterium foliorum NRRL B-24224.

The Role of Endoplasmic Reticulum Chaperones in Protein Folding and Quality Control.

Molecular chaperones assist the folding of nascent chains in the cell. Chaperones also aid in quality control decisions as persistent chaperone binding can help to sort terminal misfolded proteins for degradation. There are two major molecular chaperone families in the endoplasmic reticulum (ER) that assist proteins in reaching their native structure and evaluating the fidelity of the maturation process. The ER Hsp70 chaperone, BiP, supports adenine nucleotide-regulated binding to non-native proteins that possess exposed hydrophobic regions. In contrast, the carbohydrate-dependent chaperone system involving the membrane protein calnexin and its soluble paralogue calreticulin recognize a specific glycoform of an exposed hydrophilic protein modification for which the composition is controlled by a series of glycosidases and transferases. Here, we compare and contrast the properties, mechanisms of action and functions of these different chaperones systems that work in parallel, as well as together, to assist a large variety of substrates that traverse the eukaryotic secretory pathway.

The Blackburne-Peel Index for Determining Patellar Height Is Affected by Tibial Slope.

PURPOSE: To establish a quantitative relationship between the Blackburne-Peel index and posterior tibial slope in both skeletally mature and skeletally immature individuals and to evaluate the rate at which variation in tibial slope influences changes in patellar height categorization as normal, patella alta, and patella baja. METHODS: A consecutive series of lateral knee radiographs were retrospectively reviewed. Radiographs were excluded for rotation, inadequate visible proximal tibia length, and obstructive hardware/pathology. Modified tibial slopes of 0°, 5°, 10°, and 15° were projected anteriorly from the medial tibial plateau as described by Blackburne-Peel. The Blackburne-Peel index was determined at each modified tibial slope interval. Caton-Deschamps and Insall-Salvati indices also were measured for comparison. The rate of Blackburne-Peel index change with increase in posterior tibial slope was quantitatively analyzed. RESULTS: Fifty skeletally mature and 50 skeletally immature radiographs were included. In the skeletally mature, Blackburne-Peel indices decreased on average by 0.037, 0.044, and 0.049 as posterior tibial slope increased from 0-5°, 5-10°, and 10-15°, respectively. In the skeletally immature, Blackburne-Peel indices decreased on average by 0.045, 0.053, and 0.059 as posterior tibial slope increased from 0-5°, 5-10°, and 10-15°, respectively. Overall, 29 individuals with 0° of tibial slope were categorized as patella alta by the Blackburne-Peel index, and only 16 (55%) remained categorized as patella alta after increasing their posterior tibial slope to 15°. CONCLUSIONS: This study quantitatively demonstrates the relationship between posterior tibial slope and the Blackburne-Peel index. As expected, as posterior tibial slope increases, the Blackburne-Peel index decreases. While the change in the Blackburne-Peel index per 5° change in tibial slope appears to be small, nearly half (45%) of individuals categorized as patella alta with 0° of tibial slope were categorized as normal when their posterior tibial slope was systematically increased from 0° to 15°. When evaluating patellar height, it is important to understand how tibial slope affects the Blackburne-Peel Index measurement. CLINICAL RELEVANCE: As posterior tibial slope increases, the numerator of the Blackburne-Peel ratio decreases, and vice versa. This relationship can lead to incorrect assessment of patellar height. Objectively placing individuals into patella alta and baja categories may influence patient care and decision making.

Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway, yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from the ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells. The 71 UGGT substrates identified were mainly large multidomain and heavily glycosylated proteins when compared to the general N-glycoproteome. UGGT1 was the dominant glucosyltransferase with a preference toward large plasma membrane proteins whereas UGGT2 favored the modification of smaller, soluble lysosomal proteins. This study sheds light on differential specificities and roles of UGGT1 and UGGT2 and provides insight into the cellular reliance on the carbohydrate-dependent chaperone system to facilitate proper folding and maturation of the cellular N-glycoproteome.

Proper secretion of the serpin antithrombin relies strictly on thiol-dependent quality control.

The protein quality control machinery of the endoplasmic reticulum (ERQC) ensures that client proteins are properly folded. ERQC substrates may be recognized as nonnative by the presence of exposed hydrophobic surfaces, free thiols, or processed N-glycans. How these features dictate which ERQC pathways engage a given substrate is poorly understood. Here, using metabolic labeling, immunoprecipitations, various biochemical assays, and the human serpin antithrombin III (ATIII) as a model, we explored the role of ERQC systems in mammalian cells. Although ATIII has N-glycans and a hydrophobic core, we found that its quality control depended solely on free thiol content. Mutagenesis of all six Cys residues in ATIII to Ala resulted in its efficient secretion even though the product was not natively folded. ATIII variants with free thiols were retained in the endoplasmic reticulum but not degraded. These results provide insight into the hierarchy of ERQC systems and reveal a fundamental vulnerability of ERQC in a case of reliance on the thiol-dependent quality control pathway.

Protein Quality Control in the Endoplasmic Reticulum.

The site of protein folding and maturation for the majority of proteins that are secreted, localized to the plasma membrane or targeted to endomembrane compartments is the endoplasmic reticulum (ER). It is essential that proteins targeted to the ER are properly folded in order to carry out their function, as well as maintain protein homeostasis, as accumulation of misfolded proteins could lead to the formation of cytotoxic aggregates. Because protein folding is an error-prone process, the ER contains protein quality control networks that act to optimize proper folding and trafficking of client proteins. If a protein is unable to reach its native state, it is targeted for ER retention and subsequent degradation. The protein quality control networks of the ER that oversee this evaluation or interrogation process that decides the fate of maturing nascent chains is comprised of three general types of families: the classical chaperones, the carbohydrate-dependent system, and the thiol-dependent system. The cooperative action of these families promotes protein quality control and protein homeostasis in the ER. This review will describe the families of the ER protein quality control network and discuss the functions of individual members.

N-Glycan-based ER Molecular Chaperone and Protein Quality Control System: The Calnexin Binding Cycle.

Helenius and colleagues proposed over 20-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate-binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review.