Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is a neurometabolic disorder in the lysine metabolism pathway. In 2014 and 2021, the International PDE consortium published consensus guidelines about diagnosis and management. In this follow-on, a literature review was performed and nutrition management was evaluated through an international dietary questionnaire with 40 respondents. This manuscript discusses consensus dietary statements and the practical provision of lysine reduction therapies. Results from the questionnaire, statements from the PDE consensus guidelines, new data from the literature, as well as clinical practice experience of the metabolic dietitian group form the basis of these updated practical diet recommendations. These dietary management recommendations can support dietitians, nutritionists, and physicians in initiation and monitoring of lysine reduction therapies for PDE-ALDH7A1 patients and families.

The objective of this guide is to provide to nuclear medicine physicians a tool based on scientific evidence and prepared by consensus of experts, to perform the 18F-DCFPyL PET/CT procedure with safely and efficiently for patients with prostate cancer who present PSMA overexpression. For them, some recommendations will be established for 18F-DCFPyL PET/CT examination: reconstruction parameters, presentation of the images and their interpretation. The possible false positives of the procedure will be analysed, how to interpret them and how to avoid them. Finally, all exploration should lead to the preparation of a report that answers the clinician\'s question. For this, it is recommended to prepare a structured report that includes the PROMISE criteria as well as the classification of the findings according to PSMA-RADS parameters.

The number of publications in the field of chemical cross-linking/mass spectrometry (MS) for deriving protein 3D structures and for probing protein/protein interactions has largely increased during the last years. MS analysis of the large cross-linking data sets requires an automated data analysis by dedicated software tools, but applying scoring procedures with statistical methods does not eliminate the fundamental problems of a misassignment of cross-linked products. In fact, we have observed a significant rate of misassigned cross-links in a number of publications, mainly due to the presence of isobaric cross-linked species, an incomplete fragmentation of cross-linked products, and low-mass accuracy fragment ion data. These false assignments will eventually lead to wrong conclusions on the structural information derived from chemical cross-linking/MS experiments. In this contribution, we examine the most common sources for misassigning cross-linked products. We propose and discuss rational criteria and suggest five guidelines that might be followed for a reliable and unambiguous identification of cross-links, independent of the software used for data analysis. In the interest of the cross-linking/MS approach, it should be ensured that only high-quality data enter the structural biology literature. Clearly, there is an urgent need to define common standards for data analysis and reporting formats of cross-linked products.

Protein lysine methylation is emerging as a general post-translational modification (PTM) with essential functions regulating protein stability, activity, and protein-protein interactions. One of the outstanding challenges in this field is linking protein lysine methyltransferases (PKMTs) with specific substrates and lysine methylation events in a systematic manner. Inability to validate reported PKMT substrates delayed progress in the field and cast unnecessary doubt about protein lysine methylation as a truly general PTM. Here, we aim to provide a concise guide to help avoid some of the most common pitfalls in studies searching for new PKMT substrates and propose a set of seven basic biochemical rules: (1) include positive controls; (2) use target lysine mutations of substrate proteins as negative controls; (3) use inactive enzyme variants as negative controls; (4) report quantitative methylation data; (5) consider PKMT specificity; (6) validate methyl lysine antibodies; and (7) connect cellular and in vitro results. We explain the logic behind them and discuss how they should be implemented in the experimental work.

Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.

Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C βI (PKCβI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCβI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this \"structurally formed\" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.

TRIM5α is a type I interferon-stimulated anti-retroviral restriction factor expressed in most primates and homologous proteins are expressed in other mammals. Through its C-terminal PRYSPRY (B30.2) domain, TRIM5α binds to incoming and intact post-fusion retroviral cores in the cytoplasm. Following this direct interaction, the retroviral capsid core is destabilized and progression of the virus life cycle is interrupted. Specific recognition of its viral target by TRIM5α also triggers the induction of an antiviral state involving the activation of transcription factors NF-κB- and AP-1. In addition to PRYSPRY, several other TRIM5α domains are important for anti-retroviral function, including a RING zinc-binding motif. This domain has \"E3\" ubiquitin ligase activity and is involved in both the direct inhibition of incoming retroviruses and innate immune activation. A highly conserved sumoylation consensus site is present between the RING motif and the N-terminal extremity of TRIM5α. No clear role in restriction has been mapped to this sumoylation site, and no sumoylated forms of TRIM5α have been observed. Here we confirm that mutating the putatively sumoylated lysine (K10) of the Rhesus macaque TRIM5α (TRIM5αRh) to an arginine has only a small effect on restriction. However, we show that the mutation significantly decreases the TRIM5α-induced generation of free K63-linked ubiquitin chains, an intermediate in the activation of innate immunity pathways. Accordingly, K10R decreases TRIM5α-mediated activation of both NF-κB and AP-1. Concomitantly, we find that K10R causes a large increase in the levels of ubiquitylated TRIM5α. Finally, treatment with the nuclear export inhibitor leptomycin B shows that K10R enhances the nuclear localization of TRIM5αRh, while at the same time reducing its level of association with nuclear SUMO bodies. In conclusion, the TRIM5α sumoylation site appears to modulate the E3 ubiquitin ligase activities of the adjacent RING domain, promoting K63-linked ubiquitin chains at the expense of auto-ubiquitylation which is probably K48-linked. Consistently, we find this sumoylation site to be important for innate immune activation by TRIM5α. In addition, lysine 10 regulates TRIM5α nuclear shuttling and nuclear localization, which may also be related to its role in innate immunity activation.

Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the epsilon-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys(94) and Lys(141) of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys(94) and Lys(141), but not Lys(709) and Lys(742), resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys(94). These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites.

We report on a 4.5-year-old patient diagnosed with Glutaric aciduria type I (GAI), an autosomal recessive inborn error of lysine, hydroxylysine and tryptophan metabolism. Enzymatic assay in cultivated skin fibroblasts revealed complete absence of glutaryl-CoA dehydrogenase activity. All 11 Exons of the GCDH-Gen were sequenced and homozygosity for a yet undescribed mutation was identified. The patient was treated following the recently published guidelines for GA-I. Following this treatment regimen, the child developed normally without any manifest clinical crises. Our patient provides evidence that early commencement and strict adherence to treatment improves clinical outcome even in patients with complete absence of enzyme activity.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin ligase that regulates a diverse array of physiological processes via forming Lys-63 linked polyubiquitin chains. In this study, the lysine selection process for TRAF6/p62 ubiquitination was examined. The protein sequence of two characterized TRAF6/p62 substrates, NRIF and TrkA, revealed a conserved consensus pattern for the ubiquitination site of these two TRAF6 substrates. The consensus pattern established in the verified substrates was common to the other Trk receptor family members, TrkB and TrkC. Interestingly, Lysine 811 in TrkB was selected for ubiquination, and mutation of Lysine 811 diminished the formation of TRAF6/p62 complex that is necessary for effective ubiquination. Moreover, downstream signaling was affected upon binding of BDNF to the mutant TrkB receptor. These findings reveal a possible selection process for targeting a specific lysine residue by a single E3 ligase and underscore the role of the scaffold, p62, in this process.