Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a poorly characterized endoplasmic reticulum (ER)-localized transmembrane prolyl 4-hydroxylase (P4H-TM) is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome. Here, the 3D structure of the soluble

Alpha-catenin binds directly to beta-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates alpha-catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of alpha-T(testes)-catenin, an alpha-catenin isoform

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein that is required for successful long-term memory formation. Ca2+/CaM activates CaMKII by binding to its regulatory segment, thereby making the substrate binding pocket available. One exceptional feature of this kinase is that two binding partners have been shown persistently activate CaMKII after the Ca2+ stimulus dissipates

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells

Pathology differentiation of renal cancer types is challenging due to tissue similarities or overlapping histological features of various tumor (sub)types. As assessment is often manually conducted outcomes can be prone to human error and therefore require high-level expertise and experience. Mass spectrometry can provide detailed histo-molecular information on tissue and is becoming increasingly popular

Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergetic machine, we analyzed the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We prove that in diverse mammalian

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhanced RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing

Structures of membrane proteins are challenging to determine experimentally and currently represent only about 2% of the structures in the ProteinDataBank. Because of this disparity, methods for modeling membrane proteins are fewer and of lower quality than those for modeling soluble proteins. However, better expression, crystallization, and cryo-EM techniques have prompted a recent increase in experimental

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. A recently identified tubulin modification, methylation, occurs on mitotic spindle microtubules during cell division, and is enzymatically added to tubulin by the histone methyltransferase SETD2. We used a truncated version of human SETD2 (tSETD2) containing the catalytic SET and C-terminal

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting

Mutational effects predictions continue to improve in accuracy as advanced artificial intelligence (AI) algorithms are trained on exhaustive experimental data. The next natural questions to ask are if it is now possible to gain insights into which attribute of the mutation contributes how much to the mutational effects, and if one can develop universal rules for mapping the descriptors to mutational

In this work we present a method for the encapsulation of intracellular enzymes into a shell of albumin, using the example of catalase. We take advantage of organic, inorganic and physical chemistry for the preparation of biochemically active micrometer particles to prevent oxygen toxicity induced by artificial oxygen carriers of any type. In cell culture experiments, catalase capsules presented to

The conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding

Multi-drug efflux is a major mechanism of acquiring antimicrobial resistance among superbugs. In this study, we report the X-ray structure of NorC, a 14 transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 angstroms. The NorC structure was determined in complex with a single-domain

Background- The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested

We report the 2.4 Angstrom resolution structure of the light harvesting 2 complex (LH2) from Marichromatium (Mch.) purpuratum determined by electron cryo-microscopy. The structure contains a heptameric ring that is unique among all known LH2 structures, explaining the unusual spectroscopic properties of this bacterial antenna complex. Two sets of distinct carotenoids are identified in the structure

In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading

Epistasis is a major determinant in the emergence of novel protein function. In allosteric proteins, direct interactions between inducer-binding mutations propagate through the allosteric network, manifesting as epistasis at the level of biological function. Elucidating this relationship between local interactions and their global effects is essential to understanding evolution of allosteric proteins

An intriguing consequence of ongoing riboswitch discovery efforts is the occasional identification of metabolic or toxicity response pathways for unusual ligands. Recently, we reported the experimental validation of three distinct bacterial riboswitch classes that regulate gene expression in response to the selective binding of a guanidinium ion. These riboswitch classes, called guanidine-I, -II and

Differential scanning calorimetry and differential scanning fluorimetry were used to measure the thermal stability of human retinoid X receptor-alpha ligand binding domain (RXRα LBD) homodimer in the absence or presence of rexinoid and coactivator peptide, GRIP-1. The apo-RXRα LBD homodimer displayed a single thermal unfolding transition with a Tm of 58.7 °C and an unfolding enthalpy (ΔH) of 673 kJ/mol

The isobaric carrier approach, which combines small isobarically-labeled samples with a larger isobarically-labeled carrier sample, is finding diverse applications in ultrasensitive mass-spectrometry analysis of very small samples, such as single cells. To enhance the growing use of isobaric carriers, we characterized the trade-offs of using isobaric carriers in controlled experiments with complex

Phosphatidylinositol 3-kinase-related kinases (PIKKs) are composed of conserved FAT and kinase domains (FATKIN) along with varied solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-dependent protein kinase (DNA-PK), all existing structures represent inactive states

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the pro-apoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles

The analysis of membrane protein topography using fast photochemical oxidation of protein (FPOP) has been reported in recent years, but still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed

We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614

Colorectal cancer (CRC) is a malignant tumour with high morbidity and mortality worldwide. Efficient screening strategies for CRC and pre-cancerous lesions will promote early medical intervention and treatment, thus reducing morbidity and mortality. Proteins are generally considered key biomarkers of cancer. Herein, we performed a quantitative, tissue-original proteomics study in a cohort of ninety

Increases in soil salinity impacts growth and yield of agricultural plants by inhibiting plant functions. Soil salinization is increasing because of the pressure of a growing population on food supply. Genetically modified crops and plant breeding techniques are being used to produce plants tolerant to salt stress. However, interactions of fungal endophytes with crop plants can also improve tolerance

Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by targeting foreign DNA for destruction using CRISPR RNA-guided enzymes. CRISPR immunity begins with integration of foreign sequences into the host CRISPR genomic locus, followed by transcription and maturation of CRISPR RNAs. In a few CRISPR systems, the Cas1 integrase and a Cas6 nuclease are fused to a reverse transcriptase that

Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products (AGEs) has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devised a proteomic strategy to identify specific sites of carboxymethyllysine

Binding of dihydro-alpha-lipoic acid (DHLA) to human serum albumin (HSA) was characterised in detail in this study. Binding process was monitored by spectroscopic methods and molecular docking approach. HSA binds DHLA with moderate affinity, 0.80 ± 0.007 × 104 M-1. Spectroscopic data demonstrated that the preferential binding site for DHLA on HSA is IIA (Sudlow I). Hydrogen bonds and electrostatic

Global public health is increasingly threatened by the fast emergence of antibiotic resistance, and novel types of antibiotics are urgently needed. Metazoans have evolved their own antimicrobial mechanism, such as human group IIA secreted phospholipase A (sPLA2), which can efficiently inhibit the growth of gram-positive bacteria, but with much lower efficiency toward gram-negative bacteria. Here, we

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recently published de novo design of a four-fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities

Existing tests for detecting liver fibrosis, inflammation and steatosis, three stages of liver disease that are still reversible are severely hampered by limited accuracy or invasive nature. Here, we present a paired liver-plasma proteomics approach to infer molecular pathophysiology and to identify biomarkers in a cross-sectional alcohol-related liver disease cohort of nearly 600 individuals. Metabolic

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells through surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, we have mapped the S:ACE2 interface and uncovered long-range allosteric propagation of ACE2 binding to sites critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking

Aromatase (Cyp19) catalyzes the last biosynthetic step of estrogens in mammals and is a primary therapeutic target for postmenopausal women with hormone-related breast cancer. However, treatment with aromatase inhibitors is often associated with adverse effects and drug resistance. In this study, we used virtual screening to target a potential cytochrome P450 reductase binding site to discover four

The polyphagous pest Apolygus lucorumhas become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. The hormone 20-hydroxyecdysone (20E) regulates multiple events in insect development and physiology. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes whose expression is affected by PLC remain unknown. Here, isobaric

Global in-depth analysis of N-glycosylation, as the most complex post-translational modification of proteins, is requiring methods being as sensitive, selective and reliable as possible. Here, an enhanced strategy for N-glycomics is presented comprising optimized sample preparation yielding enhanced glycoprotein recovery and permethylation efficiency, isotopic labelling for data quality control and

Amyotrophic lateral sclerosis (ALS) is an irreversible neurodegenerative disease caused by the degeneration of motor neurons, and cytoskeletal instability is considered to be involved in neurodegeneration. A hexanucleotide repeat expansion of the C9orf72, one of the most common causes of familial ALS, produces toxic proline:arginine (PR) poly-dipeptides. PR poly-dipeptides binds polymeric forms of

Bone proteomics studies using animal proxies and skeletonized human remains have delivered encouraging results in the search for potential biomarkers for precise and accurate post-mortem interval (PMI) and the age-at-death (AAD) estimation in medico-legal investigations. At present, however, the effects of inter-individual biological differences and taphonomic alteration on recovered human bone protein

The problem of finding the configuration of points given partial information on pairwise inter-point distances, the Euclidean distance geometry problem, appears in multiple applications. In this paper, we propose an approach that integrates homology modeling and a nonconvex distance geometry algorithm for the protein structure determination problem. Preliminary numerical experiments show promising

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in

The Spike protein of SARS-CoV-2, its receptor binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans

Monolignol glucosides are storage forms of monolignols, which are polymerized to lignin to strengthen plant cell walls. The conversion of monolignol glucosides to monolignols is catalyzed by monolignol β-glucosidases. Rice Os4BGlu18 β-glucosidase catalyzes hydrolysis of the monolignol glucosides, coniferin, syringin, and p -coumaryl alcohol glucoside more efficiently than other natural substrates.

During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of

Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs and using them for diagnostics is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive assays to

Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close

DNA primase is an essential enzyme that synthesizes short RNA primers on specific DNA sequences. These RNA primers are elongated by DNA polymerase to form Okazaki fragments on the lagging DNA strand. It is therefore reasonable to as-sume that the binding of DNA primase on a genome marks the start sites of the Okazaki fragments. It has long been known that the frequency of the occurrence of primase

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

Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce)

The methylase METTL3 is the writer enzyme of the N6-methyladenosine (m6A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor (UZH1a) with potency in a biochemical assay of 280 nM, while its enantiomer UZH1b is 100 times less active. The crystal structure of the complex of METTL3 with UZH1a illustrates the interactions that make it selective against

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this

EsxA has been recognized as an important virulence factor of Mycobacterium tuberculosis (Mtb) that plays an essential role in Mtb cytosolic translocation by penetrating phagosomal membranes with its acidic pH-dependent membrane permeabilizing activity (MPA). Currently, the reported cytolytic activity of EsxA at neutral pH is controversial. In this study we have obtained direct evidence that it is the

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike

Mechanisms of amyloid inhibition remains poorly understood, in part because most protein targets of amyloid assembly are either partially unfolded or intrinsically disordered, which hinders detailed structural characterizations of protein-inhibitor complexes and structural-based mechanistic elucidation. Herein we employed a small molecule screening approach to identify inhibitors against three prototype