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The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b. [ABSTRACT FROM AUTHOR]

Abstract There is broad consensus on surgical resection being the backbone of curative therapy of gastric- and gastroesophageal junction carcinoma. Nevertheless, details on therapeutic approaches in addition to surgery, such as chemotherapy, radiotherapy or radiochemotherapy are discussed controversially; especially whether external beam radiotherapy should be applied in addition to chemotherapy and surgery is debated in both entities and differs widely between regions and centers. Early landmark trials such as the Intergroup-0116 and the MAGIC trial must be interpreted in the context of potentially insufficient lymph node resection. Despite shortcomings of both trials, benefits on overall survival by radiochemotherapy and adjuvant chemotherapy were confirmed in populations of D2-resected gastric cancer patients by Asian trials. Recent results on junctional carcinoma patients strongly suggest a survival benefit of neoadjuvant radiochemotherapy in curatively resectable patients. An effect of chemotherapy in the perioperative setting as given in the MAGIC study has been confirmed by the ACCORD07 trial for junctional carcinomas; however both the studies by Stahl et al. and the excellent outcome in the CROSS trial as compared to all other therapeutic approaches indicate a superiority of neoadjuvant radiochemotherapy as compared to perioperative chemotherapy in junctional carcinoma patients. Surgery alone without neoadjuvant or perioperative therapy is considered suboptimal in patients with locally advanced disease. In gastric carcinoma patients, perioperative chemotherapy has not been compared to adjuvant radiochemotherapy in a randomized setting. Nevertheless, the results of the recently published ARTIST trial and the Chinese data by Zhu and coworkers, indicate a superiority of adjuvant radiochemotherapy as compared to adjuvant chemotherapy in terms of disease free survival in Asian patients with advanced gastric carcinoma. The ongoing CRITICS trial is supposed to provide reliable conclusions about which therapy should be preferred in Western patients with gastric carcinoma. If radiotherapy is performed, modern approaches such as intensity-modulated radiotherapy and image guidance should be applied, as these methods reduce dose to organs at risk and provide a more homogenous coverage of planning target volumes.

Abstract Purpose Stereotactic Ablative Body Radiotherapy (SABR) is a non-invasive therapy option for inoperable liver oligometastases. Outcome and toxicity were retrospectively evaluated in a single-institution patient cohort who had undergone ultrasound-guided breath-hold SABR. Patients and methods 19 patients with liver metastases of various primary tumors consecutively treated with SABR (image-guidance with stereotactic ultrasound in combination with computer-controlled breath-hold) were analysed regarding overall-survival (OS), progression-free-survival (PFS), progression pattern, local control (LC), acute and late toxicity. Results PTV (planning target volume)-size was 108 ± 109cm3 (median 67.4 cm3). BED2 (Biologically effective dose in 2 Gy fraction) was 83.3 ± 26.2 Gy (median 78 Gy). Median follow-up and median OS were 12 months. Actuarial 2-year-OS-rate was 31%. Median PFS was 4 months, actuarial 1-year-PFS-rate was 20%. Site of first progression was predominantly distant. Regression of irradiated lesions was observed in 84% (median time to detection of regression was 2 months). Actuarial 6-month-LC-rate was 92%, 1- and 2-years-LC-rate 57%, respectively. BED2 influenced LC. When a cut-off of BED2 = 78 Gy was used, the higher BED2 values resulted in improved local control with a statistical trend to significance (p = 0.0999). Larger PTV-sizes, inversely correlated with applied dose, resulted in lower local control, also with a trend to significance (p-value = 0.08) when a volume cut-off of 67 cm3 was used. No local relapse was observed at PTV-sizes 3 and BED2 > 78 Gy. No acute clinical toxicity > °2 was observed. Late toxicity was also ≤ °2 with the exception of one gastrointestinal bleeding-episode 1 year post-SABR. A statistically significant elevation in the acute phase was observed for alkaline-phosphatase; in the chronic phase for alkaline-phosphatase, bilirubine, cholinesterase and C-reactive protein. Conclusions A trend to statistically significant correlation of local progression was observed for BED2 and PTV-size. Dose-levels BED2 > 78 Gy cannot be reached in large lesions constituting a significant fraction of this series. Image-guided SABR (igSABR) is therefore an effective non-invasive treatment modality with low toxicity in patients with small inoperable liver metastases.

Abstract Background/Purpose Radiotherapy (RT) volumes for anal cancer are large and of moderate complexity when organs at risk (OAR) such as testis, small bowel and bladder are at least partially to be shielded. Volumetric intensity modulated arc therapy (VMAT) might provide OAR-shielding comparable to step-and-shoot intensity modulated radiotherapy (IMRT) for this tumor entity with better treatment efficiency. Materials and methods Based on treatment planning CTs of 8 patients, we compared dose distributions, comformality index (CI), homogeneity index (HI), number of monitor units (MU) and treatment time (TTT) for plans generated for VMAT, 3D-CRT and step-and-shoot-IMRT (optimized based on Pencil Beam (PB) or Monte Carlo (MC) dose calculation) for typical anal cancer planning target volumes (PTV) including inguinal lymph nodes as usually treated during the first phase (0-36 Gy) of a shrinking field regimen. Results With values of 1.33 ± 0.21/1.26 ± 0.05/1.3 ± 0.02 and 1.39 ± 0.09, the CI's for IMRT (PB-Corvus/PB-Hyperion/MC-Hyperion) and VMAT are better than for 3D-CRT with 2.00 ± 0.16. The HI's for the prescribed dose (HI36) for 3D-CRT were 1.06 ± 0.01 and 1.11 ± 0.02 for VMAT, respectively and 1.15 ± 0.02/1.10 ± 0.02/1.11 ± 0.08 for IMRT (PB-Corvus/PB-Hyperion/MC-Hyperion). Mean TTT and MU's for 3D-CRT is 220s/225 ± 11MU and for IMRT (PB-Corvus/PB-Hyperion/MC-Hyperion) is 575s/1260 ± 172MU, 570s/477 ± 84MU and 610s748 ± 193MU while TTT and MU for two-arc-VMAT is 290s/268 ± 19MU. Conclusion VMAT provides treatment plans with high conformity and homogeneity equivalent to step-and-shoot-IMRT for this mono-concave treatment volume. Short treatment delivery time and low primary MU are the most important advantages.

Abstract Background Parameter optimization in the process of inverse treatment planning for intensity modulated radiation therapy (IMRT) is mainly conducted by human planners in order to create a plan with the desired dose distribution. To automate this tedious process, an artificial intelligence (AI) guided system was developed and examined. Methods The AI system can automatically accomplish the optimization process based on prior knowledge operated by several fuzzy inference systems (FIS). Prior knowledge, which was collected from human planners during their routine trial-and-error process of inverse planning, has first to be "translated" to a set of "if-then rules" for driving the FISs. To minimize subjective error which could be costly during this knowledge acquisition process, it is necessary to find a quantitative method to automatically accomplish this task. A well-developed machine learning technique, based on an adaptive neuro fuzzy inference system (ANFIS), was introduced in this study. Based on this approach, prior knowledge of a fuzzy inference system can be quickly collected from observation data (clinically used constraints). The learning capability and the accuracy of such a system were analyzed by generating multiple FIS from data collected from an AI system with known settings and rules. Results Multiple analyses showed good agreements of FIS and ANFIS according to rules (error of the output values of ANFIS based on the training data from FIS of 7.77 ± 0.02%) and membership functions (3.9%), thus suggesting that the "behavior" of an FIS can be propagated to another, based on this process. The initial experimental results on a clinical case showed that ANFIS is an effective way to build FIS from practical data, and analysis of ANFIS and FIS with clinical cases showed good planning results provided by ANFIS. OAR volumes encompassed by characteristic percentages of isodoses were reduced by a mean of between 0 and 28%. Conclusion The study demonstrated a feasible way to automatically perform parameter optimization of inverse treatment planning under guidance of prior knowledge without human intervention other than providing a set of constraints that have proven clinically useful in a given setting.

Abstract Background Image-guidance systems allow accurate interfractional repositioning of IMRT treatments, however, these may require up to 15 minutes. Therefore intrafraction motion might have an impact on treatment precision. 3D geometric data regarding intrafraction prostate motion are rare; we therefore assessed its magnitude with pre- and post-treatment fiducial-based imaging with cone-beam-CT (CBCT). Methods 39 IMRT fractions in 5 prostate cancer patients after 125I-seed implantation were evaluated. Patient position was corrected based on the 125I-seeds after pre-treatment CBCT. Immediately after treatment delivery, a second CBCT was performed. Differences in bone- and fiducial position were measured by seed-based grey-value matching. Results Fraction time was 13.6 ± 1.6 minutes. Median overall displacement vector length of 125I-seeds was 3 mm (M = 3 mm, Σ = 0.9 mm, σ = 1.7 mm; M: group systematic error, Σ: SD of systematic error, σ: SD of random error). Median displacement vector of bony structures was 1.84 mm (M = 2.9 mm, Σ = 1 mm, σ = 3.2 mm). Median displacement vector length of the prostate relative to bony structures was 1.9 mm (M = 3 mm, Σ = 1.3 mm, σ = 2.6 mm). Conclusion a) Overall displacement vector length during an IMRT session is < 3 mm. b) Positioning devices reducing intrafraction bony displacements can further reduce overall intrafraction motion. c) Intrafraction prostate motion relative to bony structures is < 2 mm and may be further reduced by institutional protocols and reduction of IMRT duration.

Abstract Background To evaluate the performance of seven different TPS (Treatment Planning Systems: Corvus, Eclipse, Hyperion, KonRad, Oncentra Masterplan, Pinnacle and PrecisePLAN) when intensity modulated (IMRT) plans are designed for paediatric tumours. Methods Datasets (CT images and volumes of interest) of four patients were used to design IMRT plans. The tumour types were: one extraosseous, intrathoracic Ewing Sarcoma; one mediastinal Rhabdomyosarcoma; one metastatic Rhabdomyosarcoma of the anus; one Wilm's tumour of the left kidney with multiple liver metastases. Prescribed doses ranged from 18 to 54.4 Gy. To minimise variability, the same beam geometry and clinical goals were imposed on all systems for every patient. Results were analysed in terms of dose distributions and dose volume histograms. Results For all patients, IMRT plans lead to acceptable treatments in terms of conformal avoidance since most of the dose objectives for Organs At Risk (OARs) were met, and the Conformity Index (averaged over all TPS and patients) ranged from 1.14 to 1.58 on primary target volumes and from 1.07 to 1.37 on boost volumes. The healthy tissue involvement was measured in terms of several parameters, and the average mean dose ranged from 4.6 to 13.7 Gy. A global scoring method was developed to evaluate plans according to their degree of success in meeting dose objectives (lower scores are better than higher ones). For OARs the range of scores was between 0.75 ± 0.15 (Eclipse) to 0.92 ± 0.18 (Pinnacle3 with physical optimisation). For target volumes, the score ranged from 0.05 ± 0.05 (Pinnacle3 with physical optimisation) to 0.16 ± 0.07 (Corvus). Conclusion A set of complex paediatric cases presented a variety of individual treatment planning challenges. Despite the large spread of results, inverse planning systems offer promising results for IMRT delivery, hence widening the treatment strategies for this very sensitive class of patients.

Abstract Background The aim of this study was to compare and to validate different dose calculation algorithms for the use in radiation therapy of small lung lesions and to optimize the treatment planning using accurate dose calculation algorithms. Methods A 9-field conformal treatment plan was generated on an inhomogeneous phantom with lung mimics and a soft tissue equivalent insert, mimicking a lung tumor. The dose distribution was calculated with the Pencil Beam and Collapsed Cone algorithms implemented in Masterplan (Nucletron) and the Monte Carlo system XVMC and validated using Gafchromic EBT films. Differences in dose distribution were evaluated. The plans were then optimized by adding segments to the outer shell of the target in order to increase the dose near the interface to the lung. Results The Pencil Beam algorithm overestimated the dose by up to 15% compared to the measurements. Collapsed Cone and Monte Carlo predicted the dose more accurately with a maximum difference of -8% and -3% respectively compared to the film. Plan optimization by adding small segments to the peripheral parts of the target, creating a 2-step fluence modulation, allowed to increase target coverage and homogeneity as compared to the uncorrected 9 field plan. Conclusion The use of forward 2-step fluence modulation in radiotherapy of small lung lesions allows the improvement of tumor coverage and dose homogeneity as compared to non-modulated treatment plans and may thus help to increase the local tumor control probability. While the Collapsed Cone algorithm is closer to measurements than the Pencil Beam algorithm, both algorithms are limited at tissue/lung interfaces, leaving Monte-Carlo the most accurate algorithm for dose prediction.

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of G q proteins by blocking GDP/GTP exchange. The inhibitor binding site of G q has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCC ICCG , an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface., Competing Interests: Declaration of interests S.G., L.P., G.A., A.M., and V.T. are employees of Carbios, and confidentiality agreements prevent them from disclosing any newly submitted declaration of invention. A.M. and V.T. have filed patents WO 2018/011284, WO 2018/011281, and WO 2020/021118 entitled “Novel esterases and uses thereof.”, (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The main protease M pro , nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with M pro . These investigations resulted in the four-armed compound 35b that binds directly to M pro . 35b could be cocrystallized with M pro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The p53 family member TAp63α plays an important role in maintaining the genetic integrity in oocytes. DNA damage, in particular DNA double strand breaks, lead to the transformation of the inhibited, only dimeric conformation into the active tetrameric one that results in the initiation of an apoptotic program. Activation requires phosphorylation by the kinase CK1 which phosphorylates TAp63α at four positions. The third phosphorylation event is the decisive step that transforms TAp63α into the active state. This third phosphorylation, however, is ~ 20 times slower than the first two phosphorylation events. This difference in the phosphorylation kinetics constitutes a safety mechanism that allows oocytes with a low degree of DNA damage to survive. So far these kinetic investigations of the phosphorylation steps have been performed with the isolated CK1 kinase domain. However, all CK1 enzymes contain C-terminal extensions that become auto-phosphorylated and inhibit the activity of the kinase. Here we have investigated the effect of auto-phosphorylation of the C-terminus in the kinase CK1δ and show that it slows down phosphorylation of the first two sites in TAp63α but basically inhibits the phosphorylation of the third site. We have identified up to ten auto-phosphorylation sites in the CK1δ C-terminal domain and show that all of them interact with the kinase domain in a "fuzzy" way in which not a single site is particularly important. Through mutation analysis we further show that hydrophobic amino acids following the phosphorylation site are important for a substrate to be able to successfully compete with the auto-inhibitory effect of the C-terminal domain. This auto-phosphorylation adds a new layer to the regulation of apoptosis in oocytes., (© 2023. Springer Nature Limited.)

Sulfonamides are one of the most important pharmacophores in medicinal chemistry, and sulfonamide analogues have gained substantial interest in recent years. However, the protein interactions of sulfonamides and especially of their analogues are underexplored. Using FKBP12 as a model system, we describe the synthesis of optically pure sulfenamide, sulfinamide, and sulfonimidamide analogues of a well characterized sulfonamide ligand. This allowed us to precisely determine the binding contributions of each sulfonamide oxygen atom and the consequences of nitrogen replacements. We also present high-resolution cocrystal structures of sulfonamide analogues buried in the pocket of a protein target. This revealed intimate contacts with the protein including an unprecedented hydrogen bond acceptor of sulfonimidamides. The use of sulfonamide analogues enabled new exit vectors that allowed remodeling of a subpocket in FKBP12. Our results illuminate the protein interaction potential of sulfonamides/sulfonamide analogues and will aid in their rational design., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15 N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative "Stablelabel" containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in "Stablelabel" lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin., (© 2023. The Author(s).)

The transcription factor p63 shares a high sequence identity with the tumour suppressor p53 which manifests itself in high structural similarity and preference for DNA sequences. Mutations in the DNA binding domain (DBD) of p53 have been studied in great detail, enabling a general mechanism-based classification. In this study we provide a detailed investigation of all currently known mutations in the p63 DBD, which are associated with developmental syndromes, by measuring their impact on transcriptional activity, DNA binding affinity, zinc binding capacity and thermodynamic stability. Some of the mutations we have further characterized with respect to their ability to convert human dermal fibroblasts into induced keratinocytes. Here we propose a classification of the p63 DBD mutations based on the four different mechanisms of DNA binding impairment which we identified: direct DNA contact, zinc finger region, H2 region, and dimer interface mutations. The data also demonstrate that, in contrast to p53 cancer mutations, no p63 mutation induces global unfolding and subsequent aggregation of the domain. The dimer interface mutations that affect the DNA binding affinity by disturbing the interaction between the individual DBDs retain partial DNA binding capacity which correlates with a milder patient phenotype., (© 2023. The Author(s).)

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Patients with myeloid neoplasia are classified by the WHO classification systems. Besides clinical and hematological criteria, cytogenetic and molecular genetic alterations highly impact treatment stratification. In routine diagnostics, a combination of methods is used to decipher different types of genetic variants. Eight patients were comprehensively analyzed using karyotyping, fluorescence in situ hybridization, array-CGH and a custom NGS panel. Clonal evolution was reconstructed manually, integrating all mutational information on single nucleotide variants (SNVs), insertions and deletions (indels), structural variants and copy number variants (CNVs). To allow a correct integration, we differentiate between three scenarios: 1) CNV occurring prior to the SNV/indel, but in the same cells. 2) SNV/indel occurring prior to the CNV, but in the same cells. 3) SNV/indel and CNV existing in parallel, independent of each other. Applying this bioinformatics approach, we reconstructed clonal evolution for all patients. This generalizable approach offers the possibility to integrate various data to analyze identification of driver and passenger mutations as well as possible targets for personalized medicine approaches. Furthermore, this model can be used to identify markers to assess the minimal residual disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sandmann, Behrens, Davenport, Thol, Heuser, Dörfel, Löhr, Castrup, Steinemann, Varghese, Schlegelberger, Dugas and Göhring.)

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5&#95;SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5&#95;SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1 H, 13 C, 15 N and 31 P resonance assignments of 5&#95;SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy., (© 2021. The Author(s).)

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7-33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1 H, 13 C and 15 N chemical shift assignment of the 29 nucleotides-RNA construct 5&#95;SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair., (© 2021. The Author(s).)

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS., (© 2021. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com , we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form., Competing Interests: CH was employed by Signals GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Altincekic, Korn, Qureshi, Dujardin, Ninot-Pedrosa, Abele, Abi Saad, Alfano, Almeida, Alshamleh, de Amorim, Anderson, Anobom, Anorma, Bains, Bax, Blackledge, Blechar, Böckmann, Brigandat, Bula, Bütikofer, Camacho-Zarco, Carlomagno, Caruso, Ceylan, Chaikuad, Chu, Cole, Crosby, de Jesus, Dhamotharan, Felli, Ferner, Fleischmann, Fogeron, Fourkiotis, Fuks, Fürtig, Gallo, Gande, Gerez, Ghosh, Gomes-Neto, Gorbatyuk, Guseva, Hacker, Häfner, Hao, Hargittay, Henzler-Wildman, Hoch, Hohmann, Hutchison, Jaudzems, Jović, Kaderli, Kalniņš, Kaņepe, Kirchdoerfer, Kirkpatrick, Knapp, Krishnathas, Kutz, zur Lage, Lambertz, Lang, Laurents, Lecoq, Linhard, Löhr, Malki, Bessa, Martin, Matzel, Maurin, McNutt, Mebus-Antunes, Meier, Meiser, Mompeán, Monaca, Montserret, Mariño Perez, Moser, Muhle-Goll, Neves-Martins, Ni, Norton-Baker, Pierattelli, Pontoriero, Pustovalova, Ohlenschläger, Orts, Da Poian, Pyper, Richter, Riek, Rienstra, Robertson, Pinheiro, Sabbatella, Salvi, Saxena, Schulte, Schiavina, Schwalbe, Silber, Almeida, Sprague-Piercy, Spyroulias, Sreeramulu, Tants, Tārs, Torres, Töws, Treviño, Trucks, Tsika, Varga, Wang, Weber, Weigand, Wiedemann, Wirmer-Bartoschek, Wirtz Martin, Zehnder, Hengesbach and Schlundt.)

The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR., (© 2020 Wiley-VCH GmbH.)

The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus' proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.