Biochemical and functional characterization of the tapasin/ERp57 conjugate

Recognition of MHC class I/peptide complexes is required for the generation of CD8+ T cell responses. Peptide loading onto MHC class I/beta2m dimers occurs in the ER and involves both specific proteins and cellular chaperones. Tapasin is essential for peptide loading onto most MHC class I alleles, and it forms a mixed disulfide with the glycoprotein specific oxidoreductase ERp57. I have characterized the biochemical requirements for tapasin/ERp57 conjugate formation and addressed potential functions for ERp57 in peptide loading. Tapasin specifically recruits ERp57 into a mixed disulfide at the expense of free ERp57 in the ER. Other components of the MHC class I peptide loading complex are not required for conjugate formation, and, in contrast to models of glycoprotein folding, conjugate formation does not require the generation of monoglucosylated glycans.Once associated, tapasin has evolved to inhibit the reductase activity of the ERp57 a domain leading to the retention of ERp57 in the loading complex over the course of normal peptide loading. In contrast, calreticulin undergoes cycles of binding and release, and its presence in the loading complex is dependent upon MHC class I. ERp57 is a core structural component of the MHC class I loading complex, and it is permanently sequestered there by tapasin.Finally, I have further characterized cells expressing a tapasin mutant unable to form the conjugate. Loading complex assembly is impaired in these cells, and peptide loading is inefficiently catalyzed by this mutant. Additionally, ERp57 binding stabilizes the structural integrity of tapasin. When endogenous ERp57 expression was suppressed using RNAi, all residual ERp57 was bound to tapasin, and both protein disulfide isomerase and ERp72, other ER resident oxidoreductases, formed mixed disulfides with tapasin. When redox mutant ERp57 proteins were over-expressed in these cells, only slight changes were seen in MHC class I trafficking. My data indicate that ERp57 is a key structural component of the MHC class I loading complex and likely does not exhibit any redox activity in this context. The key features of ERp57 responsible for its function remain unknown

TAPBPR: a new player in the MHC class I presentation pathway.

In order to provide specificity for T cell responses against pathogens and tumours, major histocompatibility complex (MHC) class I molecules present high-affinity peptides at the cell surface to T cells. A key player for peptide loading is the MHC class I-dedicated chaperone tapasin. Recently we discovered a second MHC class I-dedicated chaperone, the tapasin-related protein TAPBPR. Here, we review the major steps in the MHC class I pathway and the TAPBPR data. We discuss the potential function of TAPBPR in the MHC class I pathway and the involvement of this previously uncharacterised protein in human health and disease.C.H was supported by a Wellcome Trust PhD Studentship (Grant 089563) and L.H.B was funded by a Wellcome Trust Career Development Fellowship (Grant 085038).This is the author accepted manuscript. The final published version is available via Wiley at http://onlinelibrary.wiley.com/doi/10.1111/tan.12538/abstract;jsessionid=3D6AF64F5BD8C64E84634A4303842BE2.f04t01

Structural arrangement of the transmission interface in the antigen ABC transport complex TAP

The transporter associated with antigen processing (TAP) represents a focal point in the immune recognition of virally or malignantly transformed cells by translocating proteasomal degradation products into the endoplasmic reticulum–lumen for loading of MHC class I molecules. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex and used it to examine the interface between the transmembrane and nucleotide-binding domains (NBD) by cysteine-scanning and cross-linking approaches. Herein, we demonstrate the functional importance of the newly identified X-loop in the NBD in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in a heterodimeric ABC half-transporter complex by cysteine cross-linking. Strikingly, either substrate binding or translocation can be blocked by cross-linking the X-loop to coupling helix 2 or 1, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops and coupling helices in substrate binding, signaling, and transport

Сетевая система контроля технологического процесса выращивания полупроводниковых кристаллов и тонких пленок

Экспериментальное моделирование аппаратно-программного обеспечения показало достаточную надежность работы системы и значительное уменьшение трудоемкости контроля и управления параметрами технологического процесса

Comparison of Disk Diffusion and Etest Methods to Determine the Susceptibility of Staphylococcus aureus

Fusidic acid is a common therapy for staphylococcal infections in Saudi Arabia, but reports have suggested high rates of resistance among clinical isolates. Susceptibility testing of S. aureus to fusidic acid is further complicated by the lack of consensus on mean inhibitory concentrations (MIC) and disk diffusion cutoffs to determine resistance. The purpose of this study was to determine the correlation between disk diffusion and Etest determined MIC susceptibility results in clinical isolates of S. aureus from a large academic hospital in Riyadh, Saudi Arabia. Our data demonstrate excellent correlation between Etest determined MIC and disk diffusion susceptibility data, using either previously proposed zone sizes of ≥21 mm as susceptible and ≤18 mm as resistant or the EUCAST recommended zone size of ≤24 mm for resistance, in an area with relatively high rates of fusidic acid resistance

Rapid emergence of SARS-CoV-2 Omicron variant is associated with an infection advantage over Delta in vaccinated persons

BACKGROUND: The SARS-CoV-2 Omicron variant became a global concern due to its rapid spread and displacement of the dominant Delta variant. We hypothesized that part of Omicron’s rapid rise was based on its increased ability to cause infections in persons that are vaccinated compared to Delta. METHODS: We analyzed nasal swab PCR tests for samples collected between December 12 and 16, 2021, in Connecticut when the proportion of Delta and Omicron variants was relatively equal. We used the spike gene target failure (SGTF) to classify probable Delta and Omicron infections. We fitted an exponential curve to the estimated infections to determine the doubling times for each variant. We compared the test positivity rates for each variant by vaccination status, number of doses, and vaccine manufacturer. Generalized linear models were used to assess factors associated with odds of infection with each variant among persons testing positive for SARS-CoV-2. FINDINGS: For infections with high virus copies (Ct < 30) among vaccinated persons, we found higher odds that they were infected with Omicron compared to Delta, and that the odds increased with increased number of vaccine doses. Compared to unvaccinated persons, we found significant reduction in Delta positivity rates after two (43.4%–49.1%) and three vaccine doses (81.1%), while we only found a significant reduction in Omicron positivity rates after three doses (62.3%). CONCLUSION: The rapid rise in Omicron infections was likely driven by Omicron’s escape from vaccine-induced immunity. FUNDING: This work was supported by the Centers for Disease Control and Prevention (CDC)

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a $2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/ or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities

Tapasin's protein interactions in the rainbow trout peptide-loading complex

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.dci.2017.12.015 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Major histocompatibility complex (MHC) class I receptors play a key role in the immune system by presenting non-self peptides to T cell lymphocytes. In humans, the assembly of the MHC class I with a peptide is mediated by machinery in the endoplasmic reticulum referred as the peptide loading complex (PLC). Although, the identity of the PLC has been widely explored in humans, this complex has not been characterized in fish. Co-immunoprecipitation and mass spectrometry analysis revealed that the protein-protein interactions which exist in the human PLC are conserved in the monocyte/macrophage rainbow trout cell line (RTS11), in particular the interaction of tapasin with the transporter associated with antigen processing (TAP), MHC class I and ERp57. Importantly, a 20 kDa tapasin version that contains an intact C and N terminal domains was found to associate with ERp57 and form a 75 kDa heterodimer. These results suggest a possible novel alternative spliced version of tapasin may regulate the formation of the peptide-loading complex in teleosts.NSERC Discovery Grant number 217529-2008Canada Research Council Research Chair held by B

Accelerated SARS-CoV-2 Intrahost Evolution Leading to Distinct Genotypes During Chronic Infection

The chronic infection hypothesis for novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant emergence is increasingly gaining credence following the appearance of Omicron. Here, we investigate intrahost evolution and genetic diversity of lineage B.1.517 during a SARS-CoV-2 chronic infection lasting for 471 days (and still ongoing) with consistently recovered infectious virus and high viral genome copies. During the infection, we find an accelerated virus evolutionary rate translating to 35 nucleotide substitutions per year, approximately 2-fold higher than the global SARS-CoV-2 evolutionary rate. This intrahost evolution results in the emergence and persistence of at least three genetically distinct genotypes, suggesting the establishment of spatially structured viral populations continually reseeding different genotypes into the nasopharynx. Finally, we track the temporal dynamics of genetic diversity to identify advantageous mutations and highlight hallmark changes for chronic infection. Our findings demonstrate that untreated chronic infections accelerate SARS-CoV-2 evolution, providing an opportunity for the emergence of genetically divergent variants