Biology subjects

Schroeder, M.

Publications and source records attributed to Schroeder, M..

35 records · Page 2Linked to original sources

bioRxiv2021

Regulation of the Molecular Shuttle Service: A Multiscale Model of Aquaporin 2 Recycling

The response of cells to their environment is driven by a variety of proteins and messenger molecules. In eukaryotes their distribution, which is regulated by a vesicular transport system, is important for a tight cellular response. The recycling of aquaporin 2 between membrane and storage region is a crucial part of the body water homeostasis and its malfunction can lead to Diabetes insipidus. To understand the regulation of this system, we aggregated pathways and mechanisms from literature and derived three models in a hypothesis-driven approach. Furthermore, we combined the models to a single system to gain insight into key regulatory mechanisms. To achieve this we developed a multiscale computational framework for the modeling and simulation of cellular systems. The analysis of the system rationalises that the compartmentalization of cAMP in renal principal cells is a result of the protein kinase A signalosome and can only occur if specific cellular components are observed in conjunction. Endocytotic and exocytotic processes are inherently connected and can be regulated by the same protein kinase A signal.

systems biology↗

bioRxiv2021

Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain

The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.

biochemistry↗

bioRxiv2021

Mutations in bdcA and valS correlate with quinolone resistance in wastewater Escherichia Coli

Single mutations can confer resistance to antibiotics. Identifying such mutations can help to develop and improve drugs. Here, we systematically screen for candidate quinolone resistance-conferring mutations. We sequenced highly diverse wastewater E. coli and performed a genome-wide association study (GWAS) correlating over 200,000 mutations against quinolone resistance phenotypes. We uncovered 13 statistically significant mutations including one located at the active site of the biofilm dispersal genes bdcA and six silent mutations in the aminoacyl-tRNA synthetase valS. The study also recovered the known mutations in the topoisomerases gyrA and parC. In summary, we demonstrate that GWAS effectively and comprehensively identifies resistance mutations without a priori knowledge of targets and mode of action. The results suggest that bdcA and valS may be novel resistance genes with biofilm dispersal and translation as novel resistance mechanisms.

bioinformatics↗

bioRxiv2020

Rapid Development of Neutralizing and Diagnostic SARS-COV-2 Mouse Monoclonal Antibodies

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nanomolar-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.

immunology↗

bioRxiv2020

DFG-1 residue controls inhibitor binding mode and affinity providing a basis for rational design of kinase inhibitor selectivity

ABSTRACTSelectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, non-canonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK and CLK. Using the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a non-canonical binding mode in CLK1, providing a rational for selectivity over the closely-related CLK3 which harbors a smaller DFG-1 alanine. Our data suggests that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONSCLKCdc2 like kinasePIMProviral insertion in murine kinaseACVR1Activin receptor type IDAPKdeath-associated protein kinaseDYRKdual specificity tyrosine-phosphorylation-regulated kinasesDRAKDAP kinase-related apoptosis-inducing protein kinasesCKcasein kinaseRETProto-oncogene tyrosine-protein kinase receptor RetCDK2Cyclin-dependent kinase 2DFG motiftripeptide Asp-Phe-Gly motifVIAK motifvaline-isoleucine-alanine-lysine motif.View Full Text

biochemistry↗

bioRxiv2020

Catalytic domain plasticity of MKK7 reveals structural mechanisms of allosteric activation and new targeting opportunities

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that impact its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small molecule screening campaign yielded multiple scaffolds, including type-II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.

biochemistry↗

bioRxiv2020

Selective Vulnerability of Senescent Glioblastoma Cells to Bcl-XL Inhibition

Despite decades of research and numerous basic science advances, there have only marginal gains in improving glioblastoma multiforme survival. Therefore, new ideas and approaches for treating this aggressive disease are essential to drive progress forward. Conventional therapies, such as radiation and Temozolomide (TMZ), function to cause oxidative stress and DNA damage yielding a senescent-like state of replicative arrest in susceptible cells. However, increasing evidence demonstrates malignant cells can escape senescence leading to tumor recurrence. Ablation of non-replicating senescent tumor cells after radiation and chemotherapy may be an avenue to reduce the rates of tumor recurrence. Senolytic agents have been developed that selectively target senescent cells, but it remains unknown whether senolytics might be utilized against senescent-like glioma cells. We employed radiation or TMZ to induce a functionally senescent state in human glioblastoma cells. Viable cells that survive these treatments were then utilized to screen candidate senolytic drugs, to identify those selectively effective at ablating senescent-like cells over naive non-tumor and proliferative cells. Among 10 candidate senolytic drugs evaluated, only Bcl-XL inhibitors demonstrated reproducible senolytic activity in radiated or TMZ-treated glioma across the majority of GBM cell lines evaluated. Conversely, Bcl-2 inhibitors and other established senolytic drugs failed to show any consistent senolytic activity. In agreement with these data, Bcl-XL knockdown selectively reduced the viability of senescent-like GBM cells, whereas knockdown of Bcl-2 or Bcl-W yielded no senolytic effect. These findings demonstrate the potential to harness radiation-induced biology to ablate latent surviving cells and highlight Bcl-XL dependency as a potential vulnerability of surviving GBM cells after exposure to radiation or TMZ.

cancer biology↗

bioRxiv2020

Machine learning pattern recognition and differential network analysis of gastric microbiome in the presence of proton pump inhibitor treatment or Helicobacter pylori infection

Although long thought to be a sterile and inhospitable environment, the stomach is inhabited by diverse microbial communities, co-existing in a dynamic balance. Long-term use of orally administered drugs such as Proton Pump Inhibitors (PPIs), or bacterial infection such as Helicobacter pylori, cause significant microbial alterations. Yet, studies revealing how the commensal bacteria re-organize, due to these perturbations of the gastric environment, are in the early phase. They mainly focus on the most prevalent taxa and rely on linear techniques for multivariate analysis. Here we disclose the importance of complementing linear dimensionality reduction techniques such as Principal Component Analysis and Multidimensional Scaling with nonlinear approaches derived from the physics of complex systems. Then, we show the importance to complete multivariate pattern analysis with differential network analysis, to reveal mechanisms of re-organizations which emerge from combinatorial microbial variations induced by a medical treatment (PPIs) or an infectious state (H. pylori).

systems biology↗

bioRxiv2020

ATF4 mediates fetal globin upregulation in response to reduced β-globin

Fetal development and anemias such as {beta}-hemoglobinopathies trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal {gamma}-globin, which has suggested strategies to treat hemoglobinopathies such as thalassemia and sickle cell disease. However, the mechanisms underlying {gamma}-globin production during cellular stress are still poorly defined. Here we use CRISPR-Cas genome editing and CRISPRi transcriptional repression to model the stress caused by reduced levels of adult {beta}-globin. We find that loss of {beta}-globin is sufficient to induce widespread globin compensation, including robust re-expression of {gamma}-globin. Time-course RNA-seq of differentiating isogenic erythroid precursors identified the ATF4 transcription factor as a causal regulator of this response. ChIP-seq of multiple erythroid precursor genotypes and differentiation states revealed that {beta}-globin knockout leads to reduced engagement of ATF4 targets involved in the unfolded protein response. This ATF4 program indirectly regulates the levels of BCL11A, a key repressor of {gamma}-globin. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could be relevant for proposed gene editing strategies to treat hemoglobinopathies.

genetics↗

bioRxiv2019

Drug repositoning or target repositioning: a structural perspective of drug-target-indication relationship for available repurposed drugs.

Drug repositioning aims to find new indications for existing drugs, in order to reduce drug development cost and time. Currently, numerous successful stories of drug repositioning have been reported and many drugs are already available on the market. Although many of those cases are products of serendipitous findings, repositioning opportunities can be uncovered systematically by following either a disease-centric approach, as a result of a close relation between an old and new indication, a target-centric one, which links a known target and its established drug to a new indication, or a drug-centric approach, which connects a known drug to a new target and its associated indication. The three approaches differ in their complexity, potential, and limits, and most important the necessary starting information and computational power. Which one is predominant in current drug repositioning and what does this imply for future developments? To address these questions, we systematically evaluated over 100 drugs, 200 targets structures and over 300 indications from the Drug Repositioning Database. Each of the analysed cases has been classified based on one of the three repositioning approaches, showing that the majority, more than 60%, falls within the disease-centric definition, around 30% within the target-centric, and only less than 10% within the drug-centric. We therefore concluded that so far repositioning has mainly been disease and target repositioning and not, as drug repositioning, as expected by definition. We discuss the reasons and suggest directions to exploit the full potential of techniques useful for drug-centric in order to sustain future rationale repositioning pipelines.

bioinformatics↗

bioRxiv2019

StructureDistiller: Structural relevance scoring increases resilience of contact maps to false positive predictions

Protein folding and structure prediction are two sides of the same coin. We propose contact maps and the related techniques of constraint-based structure reconstruction as unifying aspect of both processes. The presented Structural Relevance (SR) score quantifies the contribution of individual contacts and residues to structural integrity.\n\nIt is demonstrated that entries of a contact map are not equally relevant for structural integrity. Structure prediction methods should explicitly consider the most relevant contacts for optimal performance because they effectively double resilience toward false positively predicted contacts. Furthermore, knowledge of the most relevant contacts significantly increases reconstruction fidelity on sparse contact maps by 0.4 [A].\n\nProtein folding is commonly characterized with spatial and temporal resolution: some residues are Early Folding while others are Highly Stable with respect to unfolding events. Using the proposed SR score, we demonstrate that folding initiation and structure stabilization are distinct processes.

bioinformatics↗

bioRxiv2019

p63 sets the threshold for induction of apoptosis using a kinetically encoded ‘doorbell-like’ mechanism

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63 is the key regulator of genome integrity in oocytes. After DNA damage TAp63 is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kinetics in vitro with peptides and full length protein we show that TAp63 phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63 substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

biochemistry↗

bioRxiv2019

Structure-based drug repositioning reveals ibrutinib as VEGFR2 inhibitor

Many drugs are promiscuous and bind to multiple targets. On the one hand, these targets may be linked to unwanted side effects, but on the other, they may achieve a combined desired effect (polypharmacology) or represent multiple diseases (drug repositioning). With the growth of 3D structures of drug-target complexes, it is today possible to study drug promiscuity at the structural level and to screen vast amounts of drug-target interactions to predict side effects, polypharmacological potential, and repositioning opportunities. Here, we pursue such an approach to identify drugs inactivating B-cells, whose dysregulation can function as a driver of autoimmune diseases. Screening over 500 kinases, we identified 22, whose knock out impeded the activation of B-cells. Among these 22 is the gene KDR, whose gene product VEGFR2 is a prominent cancer target with anti-VEGFR2 drugs on the market for over a decade. The main result of this paper is that structure-based drug repositioning for the identified kinase targets identified the cancer drug ibrutinib as micromolar VEGFR2 inhibitor with a very high therapeutic index in B-cell inactivation. These findings prove that ibrutinib is not only acting on the Brutons tyrosine kinase BTK, against which it was designed. Instead, it may be a polypharmacological drug, which additionally targets angiogenesis via inhibition of VEGFR2. Therefore ibrutinib carries potential to treat other VEGFR2 associated disease. Structure-based drug repositioning explains ibrutinibs anti VEGFR2 action through a specific pattern of interactions shared between BTK and VEGFR2. Overall, structure-based drug repositioning was able to predict these findings at a fraction of the time and cost of a conventional screen.

bioinformatics↗

bioRxiv2019

Characterization of Amino Acid Recognition in Aminoacyl-tRNA Synthetases

Storage and directed transfer of information is the key requirement for the development of life. Yet any information stored on our genes is useless without its correct interpretation. The genetic code defines the rule set to decode this information. Aminoacyl-tRNA synthetases are at the heart of this process. For the first time, we extensively characterize how these enzymes distinguish all natural amino acids based on the computational analysis of crystallographic structure data. The results of this meta-analysis show that the correct read-out of genetic information is a delicate interplay between the composition of the binding site, non-covalent interactions, error correction mechanisms, and steric effects.

bioinformatics↗

bioRxiv2018

Functional and Early Folding Residues are separated in proteins to increase evolvability and robustness

The three-dimensional structure of proteins captures evolutionary ancestry, and serves as starting point to understand the origin of diseases. Proteins adopt their structure autonomously by the process of protein folding. Over the last decades, the folding process of several proteins has been studied with temporal and spatial resolution which allowed the identification of so-called Early Folding Residues (EFR) in the folding process. These structurally relevant residues become affected early in the folding process and initiate the formation of secondary structure elements and guide their assembly.\n\nUsing a dataset of 30 proteins and 3,337 residues provided by the Start2Fold database, discriminative features of EFR were identified by a systematical characterization. Therefore, proteins were represented as graphs in order to analyze topological descriptors of EFR. They constitute crucial connectors of protein regions which are distant at sequence level. Especially, these residues exhibit a high number of non-covalent contacts such as hydrogen bonds and hydrophobic interactions. This tendency also manifest as energetically stable local regions in a knowledge-based potential. Conclusively, these features are not only characteristic for EFR but also differ significantly with respect to functional residues. This unveils a split between structurally and functionally relevant residues in proteins which can drastically improve their evolvability and robustness.\n\nThe characteristics of EFR cannot be attributed to trivial features such as the accessible surface area. Thus, the presented features are novel descriptors for EFR of the folding process. Potentially, these features can be used to design classifiers to predict EFR from structure or to implement structure quality assessment programs. The shown division of labor between functional and EFR has implications for the prediction of mutation effects as well as protein design and can provide insights into the evolution of proteins. Finally, EFR allow to further the understanding of the protein folding process due to their pivotal role.\n\nAuthor summaryProteins are chains of amino acids which adopt a three-dimensional structure and are then able to catalyze chemical reactions or propagate signals in organisms. Without external influence, most proteins fold into their correct structure, and a small number of Early Folding Residues (EFR) have been shown to become affected at the very start of the process. We demonstrated that these residues are located in energetically stable local conformations. EFR are in contact to many other residues of a protein and act as hubs between sequentially distant regions of a proteins. These distinct characteristics can give insights into what causes certain residues to initiate and guide the folding process. Furthermore, it can help our understanding regarding diseases such as Alzheimers or amyotrophic lateral sclerosis which are the result of protein folding gone wrong. We further found that the structurally relevant EFR are almost exclusively non-functional. Proteins separate structure and function, which increases evolvability and robustness and gives guidance for the artificial design of proteins.

bioinformatics↗

bioRxiv2017

Whole genome analysis reveals pathogenic potential of multi-drug resistant wastewater Escherichia coli

Wastewater treatment plants play an important role in the release of antibiotic resistance into the environment. It has been shown that wastewater contains multi-drug resistant Escherichia coli, but information on strain diversity is surprisingly scarce. Here we present an exceptionally large dataset on multidrug resistant Escherichia coli, originating from wastewater, over a thousand isolates were phenotypically characterized for twenty antibiotics and for 103 isolates whole genomes were sequenced. To our knowledge this is the first study documenting such a comprehensive diversity of multi-drug resistant Escherichia coli in wastewater. The genomic diversity of the isolates was unexpectedly high and contained a high number of resistance and virulence genes. To illustrate the genomic diversity of the isolates we calculated the pan genome of the wastewater Escherichia coli and found it to contain over sixteen thousand genes. To analyse this diverse dataset, we devised a computational approach correlating genotypic variation and resistance phenotype, this way we were able to identify not only known, but also candidate resistance genes. Finally, we could verify that the effluent of a wastewater treatment plant will contain multi-drug resistant Escherichia coli belonging to clinically important clonal groups.

bioinformatics↗

bioRxiv2017

Backbone brackets and arginine tweezers delineate class I and class II aminoacyl tRNA synthetases

The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes.\n\nAuthor summaryAminoacyl tRNA synthetases (aaRS) are primordial enzymes essential for interpretation and transfer of genetic information. Understanding the origin of the peculiarities observed with aaRS can explain what constituted the earliest life forms and how the genetic code was established. The increasing amount of experimentally determined three-dimensional structures of aaRS opens up new avenues for high-throughput analyses of molecular mechanisms. In this study, we present an exhaustive structural analysis of ATP binding motifs. We unveil an oppositional implementation of enzyme substrate binding in each aaRS Class. While Class I binds via interactions mediated by backbone hydrogen bonds, Class II uses a pair of arginine residues to establish salt bridges to its ATP ligand. We show how nature realized the binding of the same ligand species with completely different mechanisms. In addition, we demonstrate that sequence or even structure analysis for conserved residues may miss important functional aspects which can only be revealed by ligand interaction studies. Additionally, the placement of those key residues in the structure supports a popular hypothesis, which states that prototypic aaRS were once coded on complementary strands of the same gene.

bioinformatics↗