Synthesis and Macrodomain Binding of Gln-carba-ADPr-peptide.

Mono-ADP-ribosylation is a dynamic post-translational modification (PTM) with important roles in cell signalling. This modification occurs on a wide variety of amino acids, and one of the canonical modification sites within proteins is the side chain of glutamic acid. Given the transient nature of this modification (acylal linkage) and the high sensitivity of ADP-ribosylated glutamic acid, stabilized isosteres are required for structural and biochemical studies.

Mapping Extracellular Space Features of Viral Encephalitis to Evaluate the Proficiency of Anti-Viral Drugs.

The extracellular space (ECS) is an important barrier against viral attack on brain cells, and dynamic changes in ECS microstructure characteristics are closely related to the progression of viral encephalitis in the brain and the efficacy of antiviral drugs. However, mapping the precise morphological and rheological features of the ECS in viral encephalitis is still challenging so far. Here, a robust approach is developed using single-particle diffusional fingerprinting of quantum dots combined with machine learning to map ECS features in the brain and predict the efficacy of antiviral encephalitis drugs.

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3.

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C.

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles.

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single-molecule or single-particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during Förster resonance energy transfer (FRET) analysis in the presence of a significant donor-only fraction.

Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b.

Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine.

Multicolor 3D Orbital Tracking.

Feedback-based single-particle tracking (SPT) is a powerful technique for investigating particle behavior with very high spatiotemporal resolution. The ability to follow different species and their interactions independently adds a new dimension to the information available from SPT. However, only a few approaches have been expanded to multiple colors and no method is currently available that can follow two differently labeled biomolecules in 4 dimensions independently.

Biosynthesis of cyanobacterin, a paradigm for furanolide core structure assembly.

The γ-butyrolactone motif is found in many natural signaling molecules and other specialized metabolites. A prominent example is the potent aquatic phytotoxin cyanobacterin, which has a highly functionalized γ-butyrolactone core structure. The enzymatic machinery that assembles cyanobacterin and structurally related natural products (herein termed furanolides) has remained elusive for decades.

Design and Evaluation of TIM-3-CD28 Checkpoint Fusion Proteins to Improve Anti-CD19 CAR T-Cell Function.

Therapeutic targeting of inhibitory checkpoint molecules in combination with chimeric antigen receptor (CAR) T cells is currently investigated in a variety of clinical studies for treatment of hematologic and solid malignancies. However, the impact of co-inhibitory axes and their therapeutic implication remains understudied for the majority of acute leukemias due to their low immunogenicity/mutational load. The inhibitory exhaustion molecule TIM-3 is an important marker for the interaction of T cells with leukemic cells.

Analysis tools for single-monomer measurements of self-assembly processes.

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed.

The Neurokinin-1 Receptor Is a Target in Pediatric Rhabdoid Tumors.

Rhabdoid tumors (RT) are among the most aggressive tumors in early childhood. Overall survival remains poor, and treatment only effectively occurs at the cost of high toxicity and late adverse effects. It has been reported that the neurokinin-1 receptor/ substance P complex plays an important role in cancer and proved to be a promising target.

Zero-mode waveguides visualize the first steps during gelsolin-mediated actin filament formation.

Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level.

CAR T cell therapy in solid tumors: a short review.

Chimeric antigen receptor (CAR) T cell therapy has been established in the treatment of hematological malignancies. However, in solid tumors its efficacy remains limited. The aim of this article is to give an overview of the field of cell therapy itself, to introduce the underlying concepts of CAR T cell-based treatment approaches and to address its limitations in advancing the treatment for solid malignancies.

Structural and DNA-binding properties of the cytoplasmic domain of Vibrio cholerae transcription factor ToxR.

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae.

Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS.

The amount of 5-methyl-2'-deoxycytidine (mdC) and its oxidized derivatives 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify mdC, hmdC, fdC, and cadC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).

Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure.

SopD from Salmonella specifically inactivates Rab8.

Salmonella outer protein D (SopD) is secreted into a host during the first stages of the Salmonella infection and contributes to the systemic virulence of the bacterium. SopD2 is a SopD homolog and possesses GTPase activating protein (GAP) activity towards Rab32. Here, we identified Rab-proteins as putative SopD-targets using a yeast two-hybrid approach.

Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology.

Structural characterization of macromolecular assemblies is often limited by the transient nature of the interactions. The development of specific chemical tools to covalently tether interacting proteins to each other has played a major role in various fundamental discoveries in recent years. To this end, protein engineering techniques such as mutagenesis, incorporation of unnatural amino acids, and methods using synthetic substrate/cosubstrate derivatives were employed.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics.

Design of buried charged networks in artificial proteins.

Soluble proteins are universally packed with a hydrophobic core and a polar surface that drive the protein folding process. Yet charged networks within the central protein core are often indispensable for the biological function. Here, we show that natural buried ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif from its surroundings to gain structural stability.

Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis.

Although many components of the cell division machinery in bacteria have been identified, the mechanisms by which they work together to divide the cell remain poorly understood. Key among these components is the tubulin FtsZ, which forms a Z ring at the midcell. FtsZ recruits the other cell division proteins, collectively called the divisome, and the Z ring constricts as the cell divides.

Loss-of-function mutations in the histone methyltransferase EZH2 promote chemotherapy resistance in AML.

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine.

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I.

characterization of 3-chloro-4-hydroxybenzoic acid building block formation in ambigol biosynthesis.

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10.

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation.

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site.