ADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of Legionnaires disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. Here we took a bioinformatic approach to search the Legionella effector repertoire for additional

The plasma proteome is the ultimate target for biomarker discovery. It stores an endless amount of information on the pathophysiological status of a living organism, which is however still difficult to comprehensively access. The high structural complexity of the plasma proteome can be addressed by either a system-wide and unbiased tool such as mass spectrometry (LC-MS/MS) or a highly sensitive targeted

Sensitive assays to measure the thermostability of membrane proteins are important tools in protein purification optimisation and drug discovery. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptor 2 (CB2) as an example. This method applies the principles of Bioluminescence Resonance Energy Transfer (BRET)

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound

SARM1 is a central executor of axonal degeneration (1). Mechanistically, SARM1 contains NADase activity, which, in response to nerve injury, depletes the key cellular metabolite, NAD+ (2-5). Interestingly, SARM1 knockout mouse models do not present any apparent physiological impairment. Yet, the lack of SARM1 protects against various neuropathies (6, 7), thereby highlighting SARM1 as a likely safe

This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. Synthesis and evaluation of a first-generation cyclic peptide inhibitor reveals that the inhibitor is active against Mpro in vitro and is non-toxic

Thioredoxins (Trxs) provide electrons to essential cellular processes such as DNA synthesis. Here, we characterize human and murine Trx1 as new iron-sulfur proteins. The [2Fe-2S] cluster is complexed using cysteinyl side chains 32 and 73 in a dimeric holo-complex. Formation of the holo-dimer depends on small structural changes of the loop connecting helices three and four and is stabilized by the formation

The discovery of extremophiles has enabled development of groundbreaking biotechnology. While most of the extremophile research is focused on thermophiles, organisms that adapt to living in cold temperature, known as psychrophiles, remain under-studied. We expressed and purified DNA polymerases PIPI and PIPB from Psychromonas ingrahamii, a psychrophile that can grow below water's freezing temperature

This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition from kinetic experiments based on the standard "kobs" method. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular

The LysR-type Calvin–Benson–Bassham cycle transcriptional regulator CbbR plays an important role in CO 2 fixation in carbon metabolism in nature, which regulates the gene expression of the key enzyme RibisCO in the Calvin–Benson–Bassham (CBB) cycle. In this study, we optimized the conditions for the transformation, expression, and purification of CbbR in the model algae Nostoc sp. PCC 7120, obtained

Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50 values determined at a single point in time, because the same IC50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic

This report describes a double-exponential algebraic equation for the time course of irreversible enzyme inhibition following the two-step mechanism E + I <==> E.I ---> EI under the steady-state approximation. Under the previously invoked rapid-equilibrium approximation [Kitz & Wilson (1962) J. Biol. Chem. 237, 3245] it was assumed that the rate constant for the reversible dissociation of the initial

The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases

It has been known that SARS-CoV-2 which is considered similar to SARS-CoV invades human respiratory epithelial cells through interaction with the human angiotensin converting enzyme II (ACE2). In this work, SARS-CoV-2S-RBD and its cell receptor ACE2 were used to investigate the blocking effect and mechanism of β-chitosan to the binding of them. Besides, inhibitory effect of β-chitosan on inflammation

L-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G- protein coupled receptors (GPCR) that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely

Fluorescent proteins enable targeted visualization of biomolecules in living cells, but their maturation is oxygen-dependent and they are susceptible to aggregation and/or suffer from poor photophysical properties. Organic fluorophores are oxygen-independent with superior photophysical properties, but targeting biomolecules in vivo is challenging. Here, we introduce two oxygen-independent chemogenetic

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides

Heavy metals can selectively accumulate in certain organs and remain in them for a long time. As a result, the accumulation of metal in one or another organ may cause strongest intoxication of the animal. The purpose of the research is to study the morphological and biochemical composition of the blood of young cattle of different breeds of the dairy and dairy-and-beef direction of productivity fed

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface

The inhibition of antioxidant systems of glutathione peroxidase 4 (GPX4) or ferroptosis suppressor protein 1 (FSP1) causes iron-dependent peroxidation of polyunsaturated phospholipids that leads to cell death, a process known as ferroptosis. The mechanisms underlying iron-dependent lipid peroxidation are under active debate. Here, we report that two endoplasmic reticulum-residing oxidoreductases, NADPH-cytochrome

Serology testing for COVID-19 is highly attractive because of the relatively short diagnosis time and the ability to test for an active immune response against the SARS-CoV-2. In many types of serology tests, the sensitivity and the specificity are directly influenced by the quality of the antigens manufactured. Protein purification of these recombinantly expressed viral antigens [e.g., spike and its

One of the defining features of sexual reproduction is the recombination events that take place during meiosis I. Recombination is both evolutionarily advantageous, but also mechanistically necessary to form the crossovers that link homologous chromosomes. Meiotic recombination is initiated through the placement of programmed double-strand DNA breaks (DSBs) mediated by the protein Spo11. The timing

We reveal the structure of a type IV-B CRISPR effector (Csf) complex at 3.9 ã resolution using cryo-electron microscopy. The complex resembles the type III-A CRISPR Csm effector complex, but lacks subunits for RNA processing and target DNA cleavage, and is surprisingly assembled upon heterogeneous non-CRISPR RNA. These findings provide the first glimpse into the assembly and function of enigmatic type

Purpose: Short-TE proton MRS is used to study metabolism in the human brain. Common analysis methods model the data as linear combination of metabolite basis spectra. This large-scale multi-site study compares the levels of the four major metabolite complexes in short-TE spectra estimated by three linear-combination modelling (LCM) algorithms. Methods: 277 short-TE spectra from a recent multi-site

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives

The O-GlcNActransferase (OGT) is localized to the nucleus and cytoplasm where it regulates nucleocytoplasmic proteins by modifying serine and threonine residues with a non-extended monosaccharide, b-N-Acetyl-Glucosamine (O-GlcNAc). With thousands ofknown O-GlcNAcmodifiedproteinsbut only oneOGTencoded in the mammalian genome, a prevailing question is howOGTselects its substrates. Prior work has indicated

Metabolism is highly compartmentalized within cells, and the sub-cellular distribution of metabolites determines their use. Quantitative sub-cellular metabolomic measurements can yield crucial insights into the roles of metabolites in cellular processes. Yet, these analyses are subject to multiple confounding factors in sample preparation. We developed Stable Isotope Labeling of Essential nutrients

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of

Cryptochromes (CRYs) are evolutionarily conserved blue-light receptors that mediate various light-induced responses in bacteria, plants, and animals. Plant cryptochromes govern a variety of critical growth and developmental processes including seed germination, flowering time and entrainment of the circadian clock. CRYs photocycle involves reduction of their flavin adenine dinucleotide (FAD)-bound

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents two conformations of its Receptor Binding Domain - a receptor-accessible "up" conformation, or a receptor-inaccessible "down" conformation. Here, we report unexpected cold sensitivity of a commonly used S ectodomain construct, and resolution of this cold sensitivity in a "down" state stabilized spike. Our

The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of

In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylates two N-atoms of N-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzyme's C-terminal cupin domain, which effects the final rearrangement

C13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products, which perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains elusive. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected for their high transcript abundance in comparison with other

Carbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures

Infection with malarial parasites renders hosts more mosquito attractive than their uninfected, healthy, counterparts. One volatile organic compound, α-pinene, is associated with Plasmodium spp. infection in multiple studies and is a known mosquito attractant. However, how malarial infection results in elevated levels of host-associated α-pinene remains unclear. One study suggests that erythrocyte

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM)

Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction

The mechanistic target of rapamycin complex 1 (mTORC1) couples cell growth to nutrient, energy and growth factor availability1-3. mTORC1 is activated at the lysosomal membrane when amino acids are replete via the Rag guanosine triphosphatases (GTPases)4-6. Rags exist in two stable states, an inactive (RagA/BGDP:RagC/DGTP) and active (RagA/BGTP:RagC/DGDP) state, during low and high cellular amino acid

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. A growing connection between Siglecs and human disease motivates improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization

The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We

Doxorubicin (DOX) is a commonly employed drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of promising DOX-loaded DNA nanocarriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize

Antimicrobial resistance is a health care crisis. The resistance-conferring mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) reduces effectiveness of antifolates, e.g., trimethoprim (TMP). Although propargyl-linked antifolates (PLAs) are much more resilient than TMP towards F98Y, this substitution still vitiates their inhibition potency. Surprisingly, differences in the enantiomeric

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs reportedly work as light-driven inward H+ pumps, as xenorhodopsin. Here we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by

The extracellular matrix (ECM) protein SNED1 has been shown to promote breast cancer metastasis and control neural crest cell-specific craniofacial development, but the cellular and molecular mechanisms by which it does so remain unknown. ECM proteins exert their functions by binding to cell surface receptors, sequestering growth factors, and interacting with other ECM proteins, actions that can be

A goal of de novo protein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric

Zn2+ is an essential regulator of coagulation and its availability in plasma is fine-tuned through buffering by human serum albumin (HSA). Non-esterified fatty acids (NEFAs) transported by HSA reduce its ability to bind/buffer Zn2+. This is important as plasma NEFA levels are elevated in type-2 diabetes mellitus (T2DM) and other diseases with an increased risk of developing thrombotic complications

We describe a novel analytical method for rapid and robust identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using any LC-TOF mass spectrometer, requires no specialised knowledge of glycan analysis and makes use of the differential resolving power of reversed phase HPLC. While this separation resolves peptides with high efficiency

The spike protein of SARS-CoV-2 is known to enable viral invasion into human cells through direct binding to host receptors including ACE2. An alternate entry receptor for the virus was recently proposed to be basigin/CD147. These early studies have already prompted a clinical trial and multiple published hypotheses of the role of this host receptor in viral infection and pathogenesis. We sought to

Covid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of the SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion

AAA+ proteases, which perform regulated protein degradation in all kingdoms of life, consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX must rotate with respect

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation

Polymerase δ-interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, moonlighting protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism, DNA replication and repair, and reactive oxygen species generation. POLDIP2 is found in multiple cellular compartments, potentially shuttled depending on its role. How POLDIP2 binds to and coordinates

Hypercholesterolemia has posed a serious threat of heart diseases and stroke worldwide. Xanthine oxidase (XO), the rate-limiting enzyme in uric acid biosynthesis, is regarded as the root of reactive oxygen species (ROS) that generates atherosclerosis and cholesterol crystals. β-Hydroxy β-methylglutaryl-coenzyme A reductase (HMGR) is a rate-limiting enzyme in cholesterol biosynthesis. Although some