Distinct Autocatalytic α- N-Methylating Precursors Expand the Borosin RiPP Family of Peptide Natural Products.

Backbone N-methylations impart several favorable characteristics to peptides including increased proteolytic stability and membrane permeability. Nonetheless, amide bond N-methylations incorporated as post-translational modifications are scarce in nature and were first demonstrated in 2017 for a single set of fungal metabolites. Here we expand on our previous discovery of iterative, autocatalytic α- N-methylating precursor proteins in the borosin family of ribosomally encoded peptide natural products. We identify over 50 putative pathways in a variety of ascomycete and basidiomycete fungi and functionally validate nearly a dozen new self-α- N-methylating catalysts. Significant differences in precursor size, architecture, and core peptide properties subdivide this new peptide family into three discrete structural types. Lastly, using targeted genomics, we link the biosynthetic origins of the potent antineoplastic gymnopeptides to the borosin natural product family. This work highlights the metabolic potential of fungi for ribosomally synthesized peptide natural products.

Real-time PCR based detection of the lactase non-persistence associated genetic variant LCT-13910C>T directly from whole blood.

Primary hypolactasia is the main cause of lactose intolerance in adults. It is strongly associated with the single genetic variant LCT-13910C>T, located upstream of the lactase encoding gene. Consequently, analysis of LCT-13910C>T has been recommended as a direct genetic test for the trait. The aim of our study was to develop a TaqMan probe based real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood, circumventing DNA isolation. The LCT-13910C>T variant was determined using the DirectBlood Genotyping PCR Kit (myPOLS Biotec, Konstanz, Germany) together with an in-house TaqMan primer-probe assay. Validity and specificity of the assay was evaluated using EDTA anti-coagulated whole blood samples and corresponding DNA samples. Results from real-time PCR were compared with results obtained by Sanger sequencing from 105 blinded whole blood samples. Validity and specificity of the assay using whole blood were comparable to those using purified genomic DNA as substrate in PCR. Genetic analysis of blood samples were in complete agreement with results obtained by Sanger sequencing. In conclusion, we present a reliable real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood further facilitating diagnosis of primary hypolactasia in symptomatic patients.

Real-time PCR based HLA-B*27 screening directly in whole blood.

The linkage between the occurrence of human leucocyte antigen B*27 (HLA-B*27) and ankylosing spondylitis or other related spondyloarthritides is well documented. PCR based methods are widely used for HLA-B*27 screening. To refine HLA-B*27 testing we aimed at establishing a real-time PCR protocol to detect the HLA-B*27 allele directly in blood samples, without DNA extraction. HLA-B*27 analysis was performed by two real-time PCRs using TaqMan primer-probe assays for B*27 specific amplification of exon 2 or exon 3 of the HLA-B gene together with a mutant of Taq polymerase for direct blood PCR. Conditions for direct blood PCR were optimized and the reliability of the direct blood PCR protocol was evaluated by re-genotyping over 200 blood samples from patients who previously underwent routine DNA-based HLA-B*27 testing. Heating blood samples at 95°C for 10 minutes significantly improved PCR performance. Results from real-time PCR based HLA-B*27 testing directly in blood of over 200 patients were in 100% concordance with results obtained by routine DNA-based HLA-B*27 genotyping. In summary, we present a reliable real-time PCR protocol for HLA-B*27 screening directly in whole blood supporting fast clarification of the presence of ankylosing spondylitis or other spondyloarthritides in suspected cases.

Data on the power of high betatrophin to predict cardiovascular deaths in coronary patients.

Betatrophin is a protein which is produced by the liver and by adipose tissue. There are no clear data about serum betatrophin's cardiovascular role and it is unknown, whether betatrophin is associated with the risk of cardiovascular death. This article provides additional data on the association of betatrophin with its power to predict cardiovascular death in coronary patients. In addition, this data article demonstrates the performance of betatrophin as a biomarker using c-statistics. Analyzed data was derived from 553 coronary patients. Betatrophin was measured in serum samples and cardiovascular deaths were recorded for a median of 7.1 years. This data article is related to a research article titled "High betatrophin in coronary patients protects from cardiovascular events" [1].

High betatrophin in coronary patients protects from cardiovascular events.

BACKGROUND AND AIMS: Betatrophin, also known as angiopoietin-like protein 8 (ANGPTL8) or lipasin, is a nutritionally-regulated mammalian-specific protein secreted by the liver and adipose tissue. Many conflicting data exist with respect to its association with type 2 diabetes mellitus (T2DM), insulin resistance, and lipid markers, but no data are available on its association with cardiovascular risk. METHODS: We measured betatrophin in 553 coronary patients undergoing coronary angiography for the evaluation of established or suspected stable coronary artery disease (CAD) and prospectively recorded cardiovascular events during a follow-up of up to 8 years. RESULTS: During follow-up, 201 patients suffered a cardiovascular event and 64 died from cardiovascular causes. High betatrophin (upper tertile) was significantly and inversely associated with cardiovascular events both univariately (HR = 0.64 [95%CI 0.47-0.87], p = 0.004) and after full adjustment including the status of CAD and T2DM (adj. HR = 0.55 [95%CI 0.40-0.76], p < 0.001). The inclusion of betatrophin into a basic prediction model for the cardiovascular event risk significantly improved the model performance (NRI = 0.728, p < 0.001). CONCLUSIONS: This study is the first to show that betatrophin predicts cardiovascular events independently of conventional risk factors including the presence of CAD and T2DM.

Substrate Plasticity of a Fungal Peptide α-N-Methyltransferase.

The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.

Unrecognized High Occurrence of Genetically Confirmed Hereditary Carnitine Palmitoyltransferase II Deficiency in an Austrian Family Points to the Ongoing Underdiagnosis of the Disease.

Adult muscle carnitine palmitoyltransferase (CPT) II deficiency is a rare autosomal recessive disorder of long-chain fatty acid metabolism. It is typically associated with recurrent episodes of exercise-induced rhabdomyolysis and myoglobinuria, in most cases caused by a c.338C > T mutation in the CPT2 gene. Here we present the pedigree of one of the largest family studies of CPT II deficiency caused by the c.338C > T mutation, documented so far. The pedigree comprises 24 blood relatives of the index patient, a 32 year old female with genetically proven CPT II deficiency. In total, the mutation was detected in 20 family members, among them five homozygotes and 15 heterozygotes. Among all homozygotes, first symptoms of CPT II deficiency occurred during childhood. Additionally, two already deceased relatives of the index patient were carriers of at least one copy of the genetic variant, revealing a remarkably high prevalence of the c.338C > T mutation within the tested family. Beside the index patient, only one individual had been diagnosed with CPT II deficiency prior to this study and three cases of CPT II deficiency were newly detected by this family study, pointing to a general underdiagnosis of the disease. Therefore, this study emphasizes the need to raise awareness of CPT II deficiency for correct diagnosis and accurate management of the disease.

A molecular mechanism for the enzymatic methylation of nitrogen atoms within peptide bonds.

The peptide bond, the defining feature of proteins, governs peptide chemistry by abolishing nucleophilicity of the nitrogen. This and the planarity of the peptide bond arise from the delocalization of the lone pair of electrons on the nitrogen atom into the adjacent carbonyl. While chemical methylation of an amide bond uses a strong base to generate the imidate, OphA, the precursor protein of the fungal peptide macrocycle omphalotin A, self-hypermethylates amides at pH 7 using S-adenosyl methionine (SAM) as cofactor. The structure of OphA reveals a complex catenane-like arrangement in which the peptide substrate is clamped with its amide nitrogen aligned for nucleophilic attack on the methyl group of SAM. Biochemical data and computational modeling suggest a base-catalyzed reaction with the protein stabilizing the reaction intermediate. Backbone N-methylation of peptides enhances their protease resistance and membrane permeability, a property that holds promise for applications to medicinal chemistry.