The homodimerization domain of the Stl repressor is crucial for efficient inhibition of mycobacterial dUTPase.

The dUTPase is a key DNA repair enzyme in Mycobacterium tuberculosis, and it may serve as a novel promising anti-tuberculosis target. Stl repressor from Staphylococcus aureus was shown to bind to and inhibit dUTPases from various sources, and its expression in mycobacterial cells interfered with cell growth. To fine-tune and optimize Stl-induced inhibition of mycobacterial dUTPase, we aimed to decipher the molecular details of this interaction.

Mitochondrial Alpha-Keto Acid Dehydrogenase Complexes: Recent Developments on Structure and Function in Health and Disease.

The present work delves into the enigmatic world of mitochondrial alpha-keto acid dehydrogenase complexes discussing their metabolic significance, enzymatic operation, moonlighting activities, and pathological relevance with links to underlying structural features. This ubiquitous family of related but diverse multienzyme complexes is involved in carbohydrate metabolism (pyruvate dehydrogenase complex), the citric acid cycle (α-ketoglutarate dehydrogenase complex), and amino acid catabolism (branched-chain α-keto acid dehydrogenase complex, α-ketoadipate dehydrogenase complex); the complexes all function at strategic points and also participate in regulation in these metabolic pathways. These systems are among the largest multienzyme complexes with at times more than 100 protein chains and weights ranging up to ~10 million Daltons.

Defective function of α-ketoglutarate dehydrogenase exacerbates mitochondrial ATP deficits during complex I deficiency.

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the affected regions of the NDUFS4 KO brain.

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase.

Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the and catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised.

Probing the E1o-E2o and E1a-E2o Interactions in Binary Subcomplexes of the Human 2-Oxoglutarate Dehydrogenase and 2-Oxoadipate Dehydrogenase Complexes by Chemical Cross-Linking Mass Spectrometry and Molecular Dynamics Simulation.

The human 2-oxoglutarate dehydrogenase complex (hOGDHc) is a key enzyme in the tricarboxylic acid cycle and is one of the main regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. Evidence was obtained for formation of a hybrid complex between the hOGDHc and its homologue the 2-oxoadipate dehydrogenase complex (hOADHc) in the L-lysine metabolic pathway, suggesting a crosstalk between the two distinct pathways. Findings raised fundamental questions about the assembly of hE1a (2-oxoadipate-dependent E1 component) and hE1o (2-oxoglutarate-dependent E1) to the common hE2o core component.

Proteomic Changes of Osteoclast Differentiation in Rheumatoid and Psoriatic Arthritis Reveal Functional Differences.

Background: Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts.

Objectives: Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA.

Redox status of cysteines does not alter functional properties of human dUTPase but the Y54C mutation involved in monogenic diabetes decreases protein stability.

Recently it was proposed that the redox status of cysteines acts as a redox switch to regulate both the oligomeric status and the activity of human dUTPase. In a separate report, a human dUTPase point mutation, resulting in a tyrosine to cysteine substitution (Y54C) was identified as the monogenic cause of a rare syndrome associated with diabetes and bone marrow failure. These issues prompt a critical investigation about the potential regulatory role of cysteines in the enzyme.

Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure.

Background: The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers.

Methods: We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc.

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes.

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes.

Structure-function analyses of the G729R 2-oxoadipate dehydrogenase genetic variant associated with a disorder of l-lysine metabolism.

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in l-lysine catabolism. Genetic findings have linked mutations in the gene to several metabolic disorders. These include α-aminoadipic and α-ketoadipic aciduria (AMOXAD), a rare disorder of l-lysine, l-hydroxylysine, and l-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments.

Proteomic identification of membrane-associated placental protein 4 (MP4) as perlecan and characterization of its placental expression in normal and pathologic pregnancies.

Background: More than 50 human placental proteins were isolated and physico-chemically characterized in the 70-80s by Hans Bohn and co-workers. Many of these proteins turned to have important role in placental functions and diagnostic significance in pregnancy complications. Among these proteins was membrane-associated placental protein 4 (MP4), for which identity or function has not been identified yet.

Mass spectrometry-based analysis of macromolecular complexes of uracil-DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations.

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil-DNA glycosylase.

High sensitivity proteomics of prostate cancer tissue microarrays to discriminate between healthy and cancerous tissue.

Biopsies, in the form of tissue microarrays (TMAs) were studied to identify anomalies indicative of prostate cancer at the proteome level. TMAs offer a valuable source of well-characterized biological material. However, because of the small tissue sample size method development was essential to provide the sensitivity and reliability necessary for the analysis.

A multipronged approach unravels unprecedented protein-protein interactions in the human 2-oxoglutarate dehydrogenase multienzyme complex.

The human 2-oxoglutaric acid dehydrogenase complex (hOGDHc) plays a pivotal role in the tricarboxylic acid (TCA) cycle, and its diminished activity is associated with neurodegenerative diseases. The hOGDHc comprises three components, hE1o, hE2o, and hE3, and we recently reported functionally active E1o and E2o components, enabling studies on their assembly. No atomic-resolution structure for the hE2o component is currently available, so here we first studied the interactions in the binary subcomplexes (hE1o-hE2o, hE1o-hE3, and hE2o-hE3) to gain insight into the strength of their interactions and to identify the interaction loci in them.

Distinguishing Core and Antenna Fucosylated Glycopeptides Based on Low-Energy Tandem Mass Spectra.

A straightforward approach has been developed to distinguish core and antenna fucosylation in glycopeptides. The method does not require derivatization and can be easily adapted into a proteomics workflow. The key aspect is to use low collision energy collision-induced dissociation (CID) (on a quadrupole time-of-flight type instrument) when only single-step fragmentation processes occur.

Structural model of human dUTPase in complex with a novel proteinaceous inhibitor.

Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein Stl (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl.

Sensitive method for glycosaminoglycan analysis of tissue sections.

A simple, isocratic HPLC method based on HILIC-WAX separation, has been developed for analyzing sulfated disaccharides of glycosaminoglycans (GAGs). To our best knowledge, this is the first successful attempt using this special phase in nano-HPLC-MS analysis. Mass spectrometry was based on negative ionization, improving both sensitivity and specificity.

The Stl repressor from is an efficient inhibitor of the eukaryotic fruitfly dUTPase.

DNA metabolism and repair is vital for the maintenance of genome integrity. Specific proteinaceous inhibitors of key factors in this process have high potential for deciphering pathways of DNA metabolism and repair. The dUTPase enzyme family is responsible for guarding against erroneous uracil incorporation into DNA.

Rapamycin (mTORC1 inhibitor) reduces the production of lactate and 2-hydroxyglutarate oncometabolites in IDH1 mutant fibrosarcoma cells.

Background: Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential.

Correction: Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

[This corrects the article DOI: 10.1371/journal.pone.

Structural Characterization of Arginine Fingers: Identification of an Arginine Finger for the Pyrophosphatase dUTPases.

Arginine finger is a highly conserved and essential residue in many GTPase and AAA+ ATPase enzymes that completes the active site from a distinct protomer, forming contacts with the γ-phosphate of the nucleotide. To date, no pyrophosphatase has been identified that employs an arginine finger fulfilling all of the above properties; all essential arginine fingers are used to catalyze the cleavage of the γ-phosphate. Here, we identify and unveil the role of a conserved arginine residue in trimeric dUTPases that meets all the criteria established for arginine fingers.

Potential steps in the evolution of a fused trimeric all-β dUTPase involve a catalytically competent fused dimeric intermediate.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer-like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D.

Quantitative Comparison of Tandem Mass Spectra Obtained on Various Instruments.

The similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI), defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy-dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D "heat map", indicating which collision energy combinations result in similar spectra, and how good this agreement is.