Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells

Background

Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.

Methodology and Principal Findings

Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.

Conclusions and Significance

The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.     

Methionine oxidation and reduction in proteins

Background

Cysteine and methionine are the two sulfur containing amino acids in proteins. While the roles of protein-bound cysteinyl residues as endogenous antioxidants are well appreciated, those of methionine remain largely unexplored.

Scope

We summarize the key roles of methionine residues in proteins.

Major conclusion

Recent studies establish that cysteine and methionine have remarkably similar functions.

General significance

Both cysteine and methionine serve as important cellular antioxidants, stabilize the structure of proteins, and can act as regulatory switches through reversible oxidation and reduction. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Background

Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.

Methods

To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).

Results

DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.

Conclusions

DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.

General significance

The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.     

Detection of electrophile-sensitive proteins

Background

Redox signaling is an important emerging mechanism of cellular function. Dysfunctional redox signaling is increasingly implicated in numerous pathologies, including atherosclerosis, diabetes, and cancer. The molecular messengers in this type of signaling are reactive species which can mediate the post-translational modification of specific groups of proteins, thereby effecting functional changes in the modified proteins. Electrophilic compounds comprise one class of reactive species which can participate in redox signaling. Electrophiles modulate cell function via formation of covalent adducts with proteins, particularly cysteine residues.

Scope of review

This review will discuss the commonly used methods of detection for electrophile-sensitive proteins, and will highlight the importance of identifying these proteins for studying redox signaling and developing novel therapeutics.

Major conclusions

There are several methods which can be used to detect electrophile-sensitive proteins. These include the use of tagged model electrophiles, as well as derivatization of endogenous electrophile–protein adducts.

General significance

In order to understand the mechanisms by which electrophiles mediate redox signaling, it is necessary to identify electrophile-sensitive proteins and quantitatively assess adduct formation. Strengths and limitations of these methods will be discussed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Role of glutamine-169 in the substrate recognition of human aminopeptidase B

Background

Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.

Methods

Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.

Results

Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC₅₀ values of inhibitors that interact with the catalytic pocket of APB. The EC₅₀ value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.

Conclusion

Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.     

Ovarian cancer ascites-derived vitronectin and fibronectin: Combined purification,molecular features and effects on cell response

Background

Intra-abdominal ascites is a complication of ovarian cancers and constitutes a permissive microenvironment for metastasis. Since fibronectin and vitronectin are key actors in ovarian cancer progression, we investigated their occurrence and molecular characteristics in various ascites fluids and the influence of these ascites-derived proteins on cell behavior.

Methods

Fibronectin and vitronectin were investigated by immunoblotting within various ascites fluids. A combined affinity-based protocol was developed to purify both proteins from the same sample. Each purified protein was characterized with regard to its molecular features (molecular mass of isoforms, tryptophan intramolecular environment, hydrodynamic radii), and its influence on cell adhesion.

Results

Fibronectin and vitronectin were found in all tested ascites. Several milligrams of purified proteins were obtained from ascites of varying initial volumes. Molecular mass isoforms and conformational lability of proteins differed according to the ascites of origin. When incorporated into the cancer cell environment, ascites-derived fibronectin and vitronectin supported cell adhesion and migration with various degrees of efficiency, and induced the recruitment of integrins into focal contacts.

Conclusions

To our knowledge, this is the first combined purification of two extracellular matrix proteins from a single pathological sample containing a great variety of bioactive molecules. This study highlights that ascites-derived fibronectin and vitronectin exhibit different properties depending on the ascites.

General significance

Investigating the relationships between the molecular properties of ascites components and ovarian cancer cell phenotype according to the ascites may be critical for a better understanding of the recurrence of this lethal disease and for further biomarker identification.     

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica

Background

Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.

Methods

Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.

Results

Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.

Conclusion

Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.

General Significance

Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.     

Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases

Background

The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases.

Scope of review

In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof.

Major conclusions

Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity.

General significance

Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.     

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions)

Background

It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Methods

Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.

Results

Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.

Conclusions

The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.

General significance

Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.     

Disulfide reduction abolishes tissue factor cofactor function

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.     

Role of estrogen receptors in pro-oxidative and anti-oxidative actions of estrogens: A perspective

Background

Estrogens are steroid hormones responsible for the primary and secondary sexual characteristics in females. While pre-menopausal women use estrogens as the main constituents of contraceptive pills, post-menopausal women use the same for Hormone Replacement Therapy. Estrogens produce reactive oxygen species by increasing mitochondrial activity and redox cycling of estrogen metabolites. The phenolic hydroxyl group present at the C3 position of the A ring of estrogens can get oxidized either by accepting an electron or by losing a proton. Thus, estrogens might act as pro-oxidant in some settings, resulting in complicated non-communicable diseases, namely, cancer and cardiovascular disorders. However, in some other settings the phenolic hydroxyl group of estrogens may be responsible for the anti-oxidative beneficial functions and thus protect against cardiovascular and neurodegenerative diseases.

Scope of review

To date, no single review article has mentioned the implication of estrogen receptors in both the pro-oxidative and anti-oxidative actions of estrogens.

Major conclusion

The controversial role of estrogens as pro-oxidant or anti-oxidant is largely dependent on cell types, ratio of different types of estrogen receptors present in a particular cell and context specificity of the estrogen hormone responses. Both pro-oxidant and anti-oxidant effects of estrogens might involve different estrogen receptors that can have either genomic or non-genomic action to manifest further hormonal response.

General significance

This review highlights the role of estrogen receptors in the pro-oxidative and anti-oxidative actions of estrogens with special emphasis on neuronal cells.     

Glycan specificity of a testis-specific lectin chaperone calmegin and effects of hydrophobic interactions

Background

Testis-specific chaperone calmegin is required for the generation of normal spermatozoa. Calmegin is known to be a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin. Although functional similarity between calnexin and calmegin has been predicted, detailed information concerned with substrate recognition by calmegin, such as glycan specificity, chaperone function and binding affinity, are obscure.

Methods

In this study, biochemical properties of calmegin and calnexin were compared using synthetic glycans and glycosylated or non-glycosylated proteins as substrates.

Results

Whereas their amino acid sequences are quite similar to each other, a certain difference in secondary structures was indicated by circular dichroism (CD) spectrum. While both of them inhibited protein heat-aggregation to a similar extent, calnexin exhibited a higher ability to facilitate protein folding. Similarly to calnexin, calmegin preferentially recognizes monoglucosylated glycans such as Glc₁Man₉GlcNAc₂ (G1M9). While the surface hydrophobicity of calmegin was higher than that of calnexin, calnexin showed stronger binding to substrate. We reasoned that lectin activity, in addition to hydrophobic interaction, contributes to this strong affinity between calnexin and substrate.

Conclusions

Although their similarity in carbohydrate binding specificities is high, there seems to be some differences in the mode of substrate recognition between calmegin and calnexin.

General significance

Properties of calmegin as a lectin-chaperone were revealed in comparison with calnexin.     

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix

Background

Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood.

Methods

We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested.

Results

MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed.

Conclusions

Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins.

General significance

Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.     

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Background

Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.

Methods

Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).

Results

To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.

Conclusions

We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.

General significance

The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.     

Supernumerary proteins of mitochondrial ribosomes

Background

Messenger RNAs encoded by mitochondrial genomes are translated on mitochondrial ribosomes that have unique structure and protein composition compared to prokaryotic and cytoplasmic ribosomes. Mitochondrial ribosomes are a patchwork of core proteins that share homology with prokaryotic ribosomal proteins and new, supernumerary proteins that can be unique to different organisms. In mammals, there are specific supernumerary ribosomal proteins that are not present in other eukaryotes.

Scope of review

Here we discuss the roles of supernumerary proteins in the regulation of mitochondrial gene expression and compare them among different eukaryotic systems. Furthermore, we consider if differences in the structure and organization of mitochondrial genomes may have contributed to the acquisition of mitochondrial ribosomal proteins with new functions.

Major conclusions

The distinct and diverse compositions of mitochondrial ribosomes illustrate the high evolutionary divergence found between mitochondrial genetic systems.

General significance

Elucidating the role of the organism-specific supernumerary proteins may provide a window into the regulation of mitochondrial gene expression through evolution in response to distinct evolutionary paths taken by mitochondria in different organisms. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Activation of H-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease

Background

Glucose induces H⁺-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H⁺-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity.

Methods

We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H⁺-ATPase activation after glucose treatment.

Results

We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35–40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H⁺-ATPase complex did not undergo dissociation, and H⁺-ATPase activation was significantly delayed.

Conclusion

Our findings indicate that the mechanism of H⁺-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H⁺-ATPase complex dissociation and the activation of the enzyme.

General significance

Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H⁺-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H⁺-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.     

Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells

Background

N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway.

Scope of review

This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins.

Major conclusions

Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway.

General significance

Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.