Characterizing disease-associated changes in post-translational modifications by mass spectrometry

Introduction: Exploring post-translational modifications (PTMs) with the use of mass spectrometry (PTMomics) is a rapidly developing area, with methods for discovery/quantification being developed and advanced on a regular basis. PTMs are highly important for the regulation of protein function, interaction and activity, both in physiological and disease states. Changes in PTMs can either cause, or be the result of a disease, making them central for biomarker studies and studies of disease pathogenesis. Recently, it became possible to study multiple PTMs simultaneously from low amount of sample material, thereby increasing coverage of the PTMome obtainable from a single sample. Thus, quantitative PTMomics holds great potential to discover biomarkers from tissue and body fluids as well as elucidating disease mechanisms through characterization of signaling pathways.

Areas covered: Recent mass spectrometry-based methods for assessment of the PTMome, with focus on the most studied PTMs, are highlighted. Furthermore, both data dependent and data independent acquisition methods are evaluated. Finally, current challenges in the field are discussed.

Expert commentary: PTMomics holds great potential for clinical and biomedical research, especially with the generation of spectral libraries of peptides and PTMs from individual patients (permanent PTM maps) for use in personalized medicine.     

Novel tool in rheumatoid arthritis diagnosis—The usage of urease flap region peptidomimetics

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and non‐RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms. Thus, to better understand the role of bacteria in RA, we synthetized 6 peptidomimetics of bacterial ureases' flap region. These peptides were then used to distinguish RA patients from healthy people sera by immunoblotting. Most patients' sera were bound to peptidomimetic characteristic for Enterobacter sp. and Klebsiella sp. flap urease. We also found similarities between peptidomimetic sequence and human proteins connected with RA. This pilot study suggests that bacteria may trigger RA via mechanism of molecular mimicry of urease to host proteins and ureases flap peptidomimetics may be potential candidate as a new additional diagnostic test.     

The Root-Associated Microbial Community of the World’s Highest Growing Vascular Plants

Upward migration of plants to barren subnival areas is occurring worldwide due to raising ambient temperatures and glacial recession. In summer 2012, the presence of six vascular plants, growing in a single patch, was recorded at an unprecedented elevation of 6150 m.a.s.l. close to the summit of Mount Shukule II in the Western Himalayas (Ladakh, India). Whilst showing multiple signs of stress, all plants have managed to establish stable growth and persist for several years. To learn about the role of microbes in the process of plant upward migration, we analysed the root-associated microbial community of the plants (three individuals from each) using microscopy and tagged amplicon sequencing. No mycorrhizae were found on the roots, implying they are of little importance to the establishment and early growth of the plants. However, all roots were associated with a complex bacterial community, with richness and diversity estimates similar or even higher than the surrounding bare soil. Both soil and root-associated communities were dominated by members of the orders Sphingomonadales and Sphingobacteriales, which are typical for hot desert soils, but were different from communities of temperate subnival soils and typical rhizosphere communities. Despite taxonomic similarity on the order level, the plants harboured a unique set of highly dominant operational taxonomic units which were not found in the bare soil. These bacteria have been likely transported with the dispersing seeds and became part of the root-associated community following germination. The results indicate that developing soils act not only as a source of inoculation to plant roots but also possibly as a sink for plant-associated bacteria.     

Effects of Heart Rate Variability Biofeedback on EEG Alpha Asymmetry and Anxiety Symptoms in Male Athletes: A Pilot Study

Heart rate variability biofeedback (HRV-BFB) has been shown as useful tool to manage stress in various populations. The present study was designed to investigate whether the biofeedback-based stress management tool consisting of rhythmic breathing, actively self-generated positive emotions and a portable biofeedback device induce changes in athletes’ HRV, EEG patterns, and self-reported anxiety and self-esteem. The study involved 41 healthy male athletes, aged 16–21 (mean 18.34 ± 1.36) years. Participants were randomly divided into two groups: biofeedback and control. Athletes in the biofeedback group received HRV biofeedback training, athletes in the control group didn’t receive any intervention. During the randomized controlled trial (days 0–21), the mean anxiety score declined significantly for the intervention group (change-4 p < 0.001) but not for the control group (p = 0.817). In addition, as compared to the control, athletes in biofeedback group showed substantial and statistically significant improvement in heart rate variability indices and changes in power spectra of both theta and alpha brain waves, and alpha asymmetry. These changes suggest better self-control in the central nervous system and better flexibility of the autonomic nervous system in the group that received biofeedback training. A HRV biofeedback-based stress management tool may be beneficial for stress reduction for young male athletes.     

The CDKN2A/p16INK4a 5′UTR sequence and translational regulation: impact of novel variants predisposing to melanoma

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5′UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.‐27del23, c.‐93‐91delAGG) were classified as causal mutations (score ≥3), along with the c.‐21C>T, c.‐34G>T, and c.‐56G>T, which had already been studied by a subset of assays. The novel c.‐42T>A as well as the previously described c.‐67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.‐14C>T, c.‐20A>G, c.‐25C>T+c.‐180G>A, c.‐30G>A, c.‐40C>T, c.‐45G>A, c.‐59C>G, c.‐87T>A, c.‐252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5′UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16^INK4a translation.     

XRCC1 deficiency influences the cytotoxicity and the genomic instability induced by Me-lex,a specific inducer of N3-methyladenine

Me-lex is a sequence-specific alkylating agent synthesized to preferentially (>90%) generate N3-methyladenine (3-mA) in the minor groove of double-strand DNA, in A-T rich regions. In this paper we investigated the effect of XRCC1 deficiency in the processing of 3-mA adducts generated by Me-lex, through the molecular analysis of the Hprt mutations and the evaluation of cytogenetic end points such as sister chromatid exchanges (SCEs), micronuclei (MN) and nucleus fragmentation. EM-C11 cells, deficient in XRCC1 activity, showed a 2.5-fold higher sensitivity to the toxicity of Me-lex compared to the DNA repair proficient parental CHO-9 cells, but were not hyper mutable. The spontaneous mutation spectrum at the Hprt locus generated in EM-C11 cells revealed a high percentage of genomic deletions. After Me-lex treatment, the percentage of genomic deletions did not increase, but a class of mutations which appeared to target regulatory regions of the gene significantly increased (p = 0.0277), suggesting that non-coding Hprt genomic sequences represent a strong target for the rare mutations induced by Me-lex. The number of SCEs per chromosome increased 3-fold above background in 50 μМ Me-lex treated CHO-9 cells, while at higher Me-lex concentrations a sharp increase in the percentage of MN and fragmented nuclei was observed. In EM-C11 cells the background level of SCEs (0.939 ± 0.182) was approximately 10-fold higher than in CHO-9 (0.129 ± 0.027) and higher levels of multinucleated cells and MN were also found. In EM-C11, even low doses of Me-lex (25 μM) led to a significant increase in genomic damage. These results indicate that XRCC1 deficiency can lead to genomic instability even in the absence of an exogenous genotoxic insult and low levels of Me-lex-induced lesions, i.e., 3-mA and/or a BER intermediate, can exacerbate this instability.