Properties of MHC Class I Presented Peptides That Enhance Immunogenicity

T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.     

Identification of Key Hinge Residues Important for Nucleotide-Dependent Allostery in E. coli Hsp70/DnaK

DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaK''s nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/P_i-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/P_i was bound. Consistent with the importance of these “hinge” residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.     

PUMA: A Unified Framework for Penalized Multiple Regression Analysis of GWAS Data

Penalized Multiple Regression (PMR) can be used to discover novel disease associations in GWAS datasets. In practice, proposed PMR methods have not been able to identify well-supported associations in GWAS that are undetectable by standard association tests and thus these methods are not widely applied. Here, we present a combined algorithmic and heuristic framework for PUMA (Penalized Unified Multiple-locus Association) analysis that solves the problems of previously proposed methods including computational speed, poor performance on genome-scale simulated data, and identification of too many associations for real data to be biologically plausible. The framework includes a new minorize-maximization (MM) algorithm for generalized linear models (GLM) combined with heuristic model selection and testing methods for identification of robust associations. The PUMA framework implements the penalized maximum likelihood penalties previously proposed for GWAS analysis (i.e. Lasso, Adaptive Lasso, NEG, MCP), as well as a penalty that has not been previously applied to GWAS (i.e. LOG). Using simulations that closely mirror real GWAS data, we show that our framework has high performance and reliably increases power to detect weak associations, while existing PMR methods can perform worse than single marker testing in overall performance. To demonstrate the empirical value of PUMA, we analyzed GWAS data for type 1 diabetes, Crohns''s disease, and rheumatoid arthritis, three autoimmune diseases from the original Wellcome Trust Case Control Consortium. Our analysis replicates known associations for these diseases and we discover novel etiologically relevant susceptibility loci that are invisible to standard single marker tests, including six novel associations implicating genes involved in pancreatic function, insulin pathways and immune-cell function in type 1 diabetes; three novel associations implicating genes in pro- and anti-inflammatory pathways in Crohn''s disease; and one novel association implicating a gene involved in apoptosis pathways in rheumatoid arthritis. We provide software for applying our PUMA analysis framework.     

Recurrent Tissue-Specific mtDNA Mutations Are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.     

Reactivation of autophagy ameliorates LMNA cardiomyopathy

Mutations in the LMNA gene, which encodes lamin A and C (lamin A/C), cause a diverse spectrum of tissue-selective diseases termed laminopathies. The most prevalent form affects striated muscles as dilated cardiomyopathy with variable skeletal muscle involvement, which includes autosomal Emery-Dreifuss muscular dystrophy. Mechanisms underlying the disease pathogenesis are beginning to be understood and they point toward defects in cell signaling. We therefore assessed putative signaling defects in a mouse model carrying a point mutation in Lmna (Lmna^H222P/H222P) that faithfully recapitulates human Emery-Dreifuss muscular dystrophy. We found that AKT-mechanistic target of rapamycin (MTOR) signaling was hyperactivated in hearts of Lmna^H222P/H222P mice and that reducing MTOR activity by pharmacological intervention ameliorated cardiomyopathy. Given the central role of MTOR in regulating autophagy, we assessed fasting-induced autophagic responses and found that they were impaired in hearts of these mice. Moreover, the improved heart function associated with pharmacological blockade of MTOR was correlated with enhanced autophagy. These findings demonstrated that signaling defects that impair autophagy underlie pathogenesis of dilated cardiomyopathy arising from LMNA mutation.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

Cancer cachexia’s metabolic signature in a murine model confirms a distinct entity

Despite recent consensus definitions, lack of specific biomarkers remains a hurdle towards a more accurate and efficient diagnosis of cancer cachexia, distinguishing cachexia as a separate entity from other wasting syndromes. In a previous pilot study, we have shown that cancer-cachectic mice have a unique metabolic fingerprint with distinct glucose and lipid alterations compared to healthy controls. Further metabolomics studies were carried out to investigate differences in metabolic profiles of cancer-cachectic mice to tumor-bearing non-cachectic mice, calorie-restricted mice, and surgically treated cancer-cachectic mice. CD2F1 mice were divided into: (1) Cachexia Group received cachexia-inducing C26 undifferentiated colon carcinoma cells; (2) Tumor-Burden Group received, non-cachectic, P388 lymphoma cells; (3) Caloric-Restriction Group, remaining cancer-free, but subjected to caloric-restriction; (4) Surgery Group, similar to Cachexia Group, but tumors resected mid-experiment; and (5) Control Group aged intact. Baseline, mid-experiment and final serum samples were collected for ¹H NMR spectroscopic analysis. After data reduction, unsupervised principal component analysis and orthogonal projections to latent structures analyses demonstrate that the unique metabolic fingerprint is independent of tumor-burden and distinct from profiles of caloric-restriction and aging. Hyperlipidemia, hyperglycemia, and reduced branched-chain amino acids distinguish cachexia from other groups. Furthermore, the profile of surgically treated mice differs from that of cachectic mice, reverting to a profile more congruent with healthy controls indicating cachexia is amenable to correction where surgical cure is possible. That metabolomic analysis of murine serum is able to differentiate cachexia from tumor-burden and caloric-restriction warrants similar translational investigations in patients to explore cancer cachexia’s unique biomarkers.     

Growth Medium-Dependent Glycine Incorporation into the Peptidoglycan of Caulobacter crescentus

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.     

The Normal Limits,Subclinical Significance,Related Metabolic Derangements and Distinct Biological Effects of Body Site-Specific Adiposity in Relatively Healthy Population

Background

The accumulation of visceral adipose tissue that occurs with normal aging is associated with increased cardiovascular risks. However, the clinical significance, biological effects, and related cardiometabolic derangements of body-site specific adiposity in a relatively healthy population have not been well characterized.

Materials and Methods

In this cross-sectional study, we consecutively enrolled 608 asymptomatic subjects (mean age: 47.3 years, 27% female) from 2050 subjects undergoing an annual health survey in Taiwan. We measured pericardial (PCF) and thoracic peri-aortic (TAT) adipose tissue volumes by 16-slice multi-detector computed tomography (MDCT) (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA) and related these to clinical characteristics, body fat composition (Tanita 305 Corporation, Tokyo, Japan), coronary calcium score (CCS), serum insulin, high-sensitivity C-reactive protein (Hs-CRP) level and circulating leukocytes count. Metabolic risk was scored by Adult Treatment Panel III guidelines.

Results

TAT, PCF, and total body fat composition all increased with aging and higher metabolic scores (all p<0.05). Only TAT, however, was associated with higher circulating leukocyte counts (ß-coef.:0.24, p<0.05), serum insulin (ß-coef.:0.17, p<0.05) and high sensitivity C-reactive protein (ß-coef.:0.24, p<0.05). These relationships persisted after adjustment in multivariable models (all p<0.05). A TAT volume of 8.29 ml yielded the largest area under the receiver operating characteristic curve (AUROC: 0.79, 95%CI: 0.74–0.83) to identify metabolic syndrome. TAT but not PCF correlated with higher coronary calcium score after adjustment for clinical variables (all p<0.05).

Conclusion

In our study, we observe that age-related body-site specific accumulation of adipose tissue may have distinct biological effects. Compared to other adiposity measures, peri-aortic adiposity is more tightly associated with cardiometabolic risk profiles and subclinical atherosclerosis in a relatively healthy population.