Identification of a de novo mutation in TLK1 associated with a neurodevelopmental disorder and immunodeficiency.

Background: The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with 'Intellectual Disability, Autosomal Dominant 57' (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. Methods: A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a novel, heterozygous variant in TLK1 (c.1435C>G, p.Q479E) by genome sequencing (GS). Single cell gel electrophoresis, western blot, flow cytometry and RNA-seq were performed in patient-derived lymphoblast cell lines. In silico, biochemical and proteomic analysis were used to determine the functional impact of the p.Q479E variant and previously reported NDD-associated TLK1 variant, p.M566T. Results: Transcriptome sequencing in patient-derived cells confirmed expression of TLK1 transcripts carrying the p.Q479E variant and revealed alterations in genes involved in class switch recombination and cytokine signaling. Cells expressing the p.Q479E variant exhibited reduced cytokine responses and higher levels of spontaneous DNA damage but not increased sensitivity to radiation or DNA repair defects. The p.Q479E and p.M566T variants impaired kinase activity but did not strongly alter localization or proximal protein interactions. Conclusion: Our study provides the first functional characterization of TLK1 variants associated with NDDs and suggests potential involvement in central nervous system and immune system development. Our results indicate that, like TLK2 variants, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation.

BACKGROUND: Vertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3' UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date. RESULTS: Based on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2-4 are regulated by multiple proline-directed phosphorylations that control their liquid-liquid phase separation. CPEB2-4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs. CONCLUSIONS: Altogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors.

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

Biomarker candidates for progression and clinical management of COVID-19 associated pneumonia at time of admission.

COVID-19 pathophysiology is currently not fully understood, reliable prognostic factors remain elusive, and few specific therapeutic strategies have been proposed. In this scenario, availability of biomarkers is a priority. MS-based Proteomics techniques were used to profile the proteome of 81 plasma samples extracted in four consecutive days from 23 hospitalized COVID-19 associated pneumonia patients. Samples from 10 subjects that reached a critical condition during their hospital stay and 10 matched non-severe controls were drawn before the administration of any COVID-19 specific treatment and used to identify potential biomarkers of COVID-19 prognosis. Additionally, we compared the proteome of five patients before and after glucocorticoids and tocilizumab treatment, to assess the changes induced by the therapy on our selected candidates. Forty-two proteins were differentially expressed between patients' evolution groups at 10% FDR. Twelve proteins showed lower levels in critical patients (fold-changes 1.20-3.58), of which OAS3 and COG5 found their expression increased after COVID-19 specific therapy. Most of the 30 proteins over-expressed in critical patients (fold-changes 1.17-4.43) were linked to inflammation, coagulation, lipids metabolism, complement or immunoglobulins, and a third of them decreased their expression after treatment. We propose a set of candidate proteins for biomarkers of COVID-19 prognosis at the time of hospital admission. The study design employed is distinctive from previous works and aimed to optimize the chances of the candidates to be validated in confirmatory studies and, eventually, to play a useful role in the clinical practice.

Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis.

INTRODUCTION: The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with 'Mental Retardation Autosomal Dominant 57' (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies. METHODS: We re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity. RESULTS: We identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage. CONCLUSION: Our study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Furan warheads for covalent trapping of weak protein-protein interactions: cross-linking of thymosin ß4 to actin.

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin ß4 (Tß4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tß4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.

Protamine Characterization by Top-Down Proteomics: Boosting Proteoform Identification with DBSCAN.

Protamines replace histones as the main nuclear protein in the sperm cells of many species and play a crucial role in compacting the paternal genome. Human spermatozoa contain protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or the P1/P2 ratio may be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteoforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down proteomics permits a more in-depth analysis of protamine PTMs and proteoforms, thereby opening up new perspectives to unravel their impact on male fertility. We report on the analysis of two normozoospermic semen samples by top-down proteomics. We discuss the difficulties encountered with the data analysis and propose solutions as this step is one of the current bottlenecks in top-down proteomics with the bioinformatics tools currently available. Our strategy for the data analysis combines two software packages, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of normozoospermic individuals represents the first step towards the future study of sperm pathological conditions opening up the potential personalized diagnosis of male infertility.

Characterization of Human Sperm Protamine Proteoforms through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches.

Protamine 1 (P1) and protamine 2 (P2) family are extremely basic, sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated in the UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

Top-Down Proteomics Applied to Human Cerebrospinal Fluid.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

Ex vivo assessment and in vivo validation of non-invasive stent monitoring techniques based on microwave spectrometry.

Some conditions are well known to be directly associated with stent failure, including in-stent re-occlusion and stent fracture. Currently, identification of these high-risk conditions requires invasive and complex procedures. This study aims to assess microwave spectrometry (MWS) for monitoring stents non-invasively. Preliminary ex vivo data are presented to move to in vivo validation. Fifteen mice were assigned to receive subcutaneous stent implantations (n = 10) or sham operations (n = 5). MWS measurements were carried out at 0, 2, 4, 7, 14, 22, and 29 days of follow-up. Additionally, 5 stented animals were summited to micro-CT analyses at the same time points. At 29 days, 3 animals were included into a stent fracture subgroup and underwent a last MWS and micro-CT analysis. MWS was able to identify stent position and in-stent stenosis over time, also discerning significant differences from baseline measures (P < 0.001). Moreover, MWS identified fractured vs. non-fractured stents in vivo. Taken together, MWS emerges as a non-invasive, non-ionizing alternative for stent monitoring. MWS analysis clearly distinguished between in-stent stenosis and stent fracture phenomena.

Microwave spectrometry for the evaluation of the structural integrity of metallic stents.

PURPOSE: To assess the feasibility of a method based on microwave spectrometry to detect structural distortions of metallic stents in open air conditions and envisage the prospects of this approach toward possible medical applicability for the evaluation of implanted stents. METHODS: Microwave absorbance spectra between 2.0 and 18.0 GHz were acquired in open air for the characterization of a set of commercial stents using a specifically design setup. Rotating each sample over 360°, 2D absorbance diagrams were generated as a function of frequency and rotation angle. To check our approach for detecting changes in stent length (fracture) and diameter (recoil), two specific tests were performed in open air. Finally, with a few adjustments, this same system provides 2D absorbance diagrams of stents immersed in a water-based phantom, this time over a bandwidth ranging from 0.2 to 1.8 GHz. RESULTS: The authors show that metallic stents exhibit characteristic resonant frequencies in their microwave absorbance spectra in open air which depend on their length and, as a result, may reflect the occurrence of structural distortions. These resonances can be understood considering that such devices behave like dipole antennas in terms of microwave scattering. From fracture tests, the authors infer that microwave spectrometry provides signs of presence of Type I to Type IV stent fractures and allows in particular a quantitative evaluation of Type III and Type IV fractures. Recoil tests show that microwave spectrometry seems able to provide some quantitative assessment of diametrical shrinkage, but only if it involves longitudinal shortening. Finally, the authors observe that the resonant frequencies of stents placed inside the phantom shift down with respect to the corresponding open air frequencies, as it should be expected considering the increase of dielectric permittivity from air to water. CONCLUSIONS: The evaluation of stent resonant frequencies provided by microwave spectrometry allows detection and some quantitative assessment of stent fracture and recoil in open air conditions. Resonances of stents immersed in water can be also detected and their characteristic frequencies are in good agreement with theoretical estimates. Although these are promising results, further verification in a more relevant phantom is required in order to foresee the real potential of this approach.