The main targets for the immunosuppressive calcineurin inhibitors, cyclosporin A (CsA) and tacrolimus, have been considered to be activated T cells, but not antigen-presenting cells. Here we demonstrate that CsA and tacrolimus, but not rapamycin, inhibit major histocompatibility complex (MHC)-restricted antigen presentation in dendritic cells (DCs). Microencapsulated ovalbumin (OVA) was efficiently captured, processed, and presented on both class I MHC molecules IntroductionDendritic cells (DCs) play a key role in the initiation of primary immune responses. 1 DCs can acquire and process antigens in the periphery, and migrate to secondary lymphoid tissues where they prime primary T-cell responses. Two distinct pathways have been known for the presentation of antigenic peptides on major histocompatibility complex (MHC) molecules. 2 Exogenous antigens internalized by phagocytosis or endocytosis are normally processed and loaded on class II MHC molecules in a post-Golgi compartment. In contrast, endogenous antigens are partially digested by proteosome, transferred into endoplasmic reticulum (ER) by a transporter associated with antigen presentation (TAP), and then loaded on class I MHC molecules. It is also known that DCs can uptake antigens from cells undergoing apoptosis or necrosis for presentation to class I MHC-restricted cytotoxic T lymphocytes (CTLs). This form of antigen presentation has been termed "cross-presentation." Crosspresentation has been demonstrated to occur for many viral antigens and also for tumor antigens. 3,4 Cross-presentation may be a mechanism by which naive T cells can be primed to antigens that are present in nonprofessional antigen-presenting cells (APCs). In the absence of such a mechanism, viral or tumor antigens expressed in nonprofessional APCs could escape immunosurveillance because CTL responses can only be induced efficiently for the antigens presented via class I MHC molecules on professional APCs. [5][6][7] Thus far, the main cellular targets for CsA and tacrolimus have been considered to be activated T cells, but not APCs. 8 Even though chemically unrelated, CsA and tacrolimus bind to and inhibit the same protein, calcineurin, in a calcium-dependent signaling pathway after formation of a complex with cyclophilin A and FK-506 binding proteins (FKBPs), respectively, resulting in inhibition of the transcription of interleukin 2 (IL-2) and other lymphokines. 9,10 Rapamycin has structural similarity with tacrolimus and even forms a complex with the same FKBPs that form a complex with tacrolimus. 11 However, the FKBP-rapamycin complex interacts with a protein distinct from calcineurin, termed the mammalian target of rapamycin, mTOR. 12 Rapamycin suppresses T-cell activation at a different level, mainly through inhibition of cell-cycle progression via suppression of p70 S6 kinase activation induced by growthpromoting lymphokines. 8 Because T-cell responses can only be initiated on recognition of peptide-MHC complexes presented by professional APCs, inhibitors of antigen processing p...

The pathogenic consequences of 369 unique human HsMLH1 missense variants has been hampered by the lack of a detailed function in mismatch repair (MMR). Here single-molecule images show that HsMSH2-HsMSH6 provides a platform for HsMLH1-HsPMS2 to form a stable sliding clamp on mismatched DNA. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of clinically relevant HsMLH1 missense mutations.

OBJECTIVE:This study aimed to identify novel GATA5 mutations that underlie familial atrial fibrillation.METHODS:A total of 110 unrelated patients with familial atrial fibrillation and 200 unrelated, ethnically matched healthy controls were recruited. The entire coding region of the GATA5 gene was sequenced in 110 atrial fibrillation probands. The available relatives of the mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional effect of the mutated GATA5 was characterized using a luciferase reporter assay system.RESULTS:Two novel heterozygous GATA5 mutations (p.Y138F and p.C210G) were identified in two of the 110 unrelated atrial fibrillation families. These missense mutations cosegregated with AF in the families and were absent in the 400 control chromosomes. A cross-species alignment of GATA5 protein sequence showed that the altered amino acids were completely conserved evolutionarily. A functional analysis revealed that the mutant GATA5 proteins were associated with significantly decreased transcriptional activation when compared with their wild-type counterpart.CONCLUSION:The findings expand the spectrum of GATA5 mutations linked to AF and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation, suggesting potential implications for the early prophylaxis and personalized treatment of this common arrhythmia.

Lynch Syndrome (LS), the most common inherited colorectal cancer predisposition, is primarily caused by mutations in the DNA mismatch repair (MMR) genes: HsMSH2, HsMLH1, HsMSH6 and HsPMS2. MMR proteins maintain genomic stability by repairing nucleotide mispairing errors during DNA replication. They are highly conserved members of the MutS (MSH) and MutL (MLH/PMS) proteins that play central roles in directing accurate MMR excision. MSH proteins recognize mismatched nucleotides which triggers ATP binding and formation of a sliding clamp that randomly diffuses along the DNA. Previous biophysical studies in E.coli, showed that EcMutS and EcMutL formed cascading sliding clamps on the mismatched DNA, and defined the intermediary activity of EcMutL, as a signal transmitter between the mismatch recognition and the downstream excision processes. However, the detailed mechanics of the human MLH/PMS proteins has remained a puzzle. Using real‐time‐single molecule imaging we show that human heterodimeric MMR proteins HsMSH2‐HsMSH6 and HsMLH1‐HsPMS2 exhibit identical mechanism to their bacterial homologs. HsMSH2‐HsMSH6 is required to load the major human MLH/PMS heterodimer onto a DNA containing a single mismatch. In its absence, no visible HsMLH1‐HsPMS2 sliding clamps were observed. MLH/PMS proteins contain a disordered region, variable in length, that connects the N‐terminal ATP binding domain that interacts with HsMSH2‐HsMSH6 with the C‐terminal dimerization domain of the protein. Our results indicate that HsMSH2‐HsMSH6 provides a platform for HsMLH1‐HsPMS2 to wrap around the DNA and form a stable sliding clamp, diffusing rapidly along the DNA. Furthermore, it has been shown that increasing the number of amino acids deleted in the linker domain in EcMutL and S. cerevisae ScMlh1 or ScPms1, initially block the ability to transit roadblocks on the DNA to ultimately, total MMR inhibition. MLH/PMS sliding clamps resemble a flexible donut that encircles the DNA with a relatively large donut hole. We hypothesize that any sequence of amino acids could, in principle, occupy this domain with the condition that it remained malleable and capable of wrapping around the DNA. Therefore, few if any LS pathogenic missense protein variants (MPVs) are expected within the HsMLH1 linker domain. To test this, unique missense protein variants (MPVs) located in the coding sequence of HsMLH1 were analyzed, A clear pattern was found, where non‐pathogenic (26) and uncertain (366) variants were distributed ubiquitously. However, the disordered linker region did not contain any pathogenic (71) variants. We conclude that uncertain MPVs located in the linker are unlikely to be pathogenic since it is doubtful that any single amino acid substitution could influence the natural disorder that is essential for linker‐domain function. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of pathogenic HsMLH1 missense mutations.