Mechanistic insights into sulfur source-driven physiological responses and metabolic reorganization in the fuel-biodesulfurizing Rhodococcus qingshengii IGTS8.

Comparative proteomics and untargeted metabolomics were combined to study the physiological and metabolic adaptations of Rhodococcus qingshengii IGTS8 under biodesulfurization conditions. After growth in a chemically defined medium with either dibenzothiophene (DBT) or MgSO4 as the sulfur source, many differentially produced proteins and metabolites associated with several metabolic and physiological processes were detected including the metabolism of carbohydrates, amino acids, lipids, nucleotides, vitamins, protein synthesis, transcriptional regulation, cell envelope biogenesis, and cell division. Increased production of the redox cofactor mycofactocin and associated proteins was one of the most striking adaptations under biodesulfurization conditions. While most central metabolic enzymes were less abundant in the presence of DBT, a key enzyme of the glyoxylate shunt, isocitrate lyase, was up to 26-fold more abundant. Several C1 metabolism and oligotrophy-related enzymes were significantly more abundant in the biodesulfurizing culture. R. qingshengii IGTS8 exhibited oligotrophic growth in liquid and solid media under carbon starvation. Moreover, the oligotrophic growth was faster on the solid medium in the presence of DBT compared to MgSO4 cultures. In the DBT culture, the cell envelope and phospholipids were remodeled, with lower levels of phosphatidylethanolamine and unsaturated and short-chain fatty acids being the most prominent changes. Biodesulfurization increased the biosynthesis of osmoprotectants (ectoine and mannosylglycerate) as well as glutamate and induced the stringent response. Our findings reveal highly diverse and overlapping stress responses that could protect the biodesulfurizing culture not only from the associated sulfate limitation but also from chemical, oxidative, and osmotic stress, allowing efficient resource management. IMPORTANCE Despite decades of research, a commercially viable bioprocess for fuel desulfurization has not been developed yet. This is mainly due to lack of knowledge of the physiology and metabolism of fuel-biodesulfurizing bacteria. Being a stressful condition, biodesulfurization could provoke several stress responses that are not understood. This is particularly important because a thorough understanding of the microbial stress response is essential for the development of environmentally friendly and industrially efficient microbial biocatalysts. Our comparative systems biology studies provide a mechanistic understanding of the biology of biodesulfurization, which is crucial for informed developments through the rational design of recombinant biodesulfurizers and optimization of the bioprocess conditions. Our findings enhance the understanding of the physiology, metabolism, and stress response not only in biodesulfurizing bacteria but also in rhodococci, a precious group of biotechnologically important bacteria.

Biodesulfurization Induces Reprogramming of Sulfur Metabolism in Rhodococcus qingshengii IGTS8: Proteomics and Untargeted Metabolomics.

Sulfur metabolism in fuel-biodesulfurizing bacteria and the underlying physiological adaptations are not understood, which has impeded the development of a commercially viable bioprocess for fuel desulfurization. To fill these knowledge gaps, we performed comparative proteomics and untargeted metabolomics in cultures of the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 grown on either inorganic sulfate or the diesel-borne organosulfur compound dibenzothiophene as a sole sulfur source. Dibenzothiophene significantly altered the biosynthesis of many sulfur metabolism proteins and metabolites in a growth phase-dependent manner, which enabled us to reconstruct the first experimental model for sulfur metabolism in a fuel-biodesulfurizing bacterium. All key pathways related to assimilatory sulfur metabolism were represented in the sulfur proteome, including uptake of the sulfur sources, sulfur acquisition, and assimilatory sulfate reduction, in addition to biosynthesis of key sulfur-containing metabolites such as S-adenosylmethionine, coenzyme A, biotin, thiamin, molybdenum cofactor, mycothiol, and ergothioneine (low-molecular weight thiols). Fifty-two proteins exhibited significantly different abundance during at least one growth phase. Sixteen proteins were uniquely detected and 47 proteins were significantly more abundant in the dibenzothiophene culture during at least one growth phase. The sulfate-free dibenzothiophene-containing culture reacted to sulfate starvation by restricting sulfur assimilation, enforcing sulfur-sparing, and maintaining redox homeostasis. Biodesulfurization triggered alternative pathways for sulfur assimilation different from those operating in the inorganic sulfate culture. Sulfur metabolism reprogramming and metabolic switches in the dibenzothiophene culture were manifested in limiting sulfite reduction and biosynthesis of cysteine, while boosting the production of methionine via the cobalamin-independent pathway, as well as the biosynthesis of the redox buffers mycothiol and ergothioneine. The omics data underscore the key role of sulfur metabolism in shaping the biodesulfurization phenotype and highlight potential targets for improving the biodesulfurization catalytic activity via metabolic engineering. IMPORTANCE For many decades, research on biodesulfurization of fossil fuels was conducted amid a large gap in knowledge of sulfur metabolism and its regulation in fuel-biodesulfurizing bacteria, which has impeded the development of a commercially viable bioprocess. In addition, lack of understanding of biodesulfurization-associated metabolic and physiological adaptations prohibited the development of efficient biodesulfurizers. Our integrated omics-based findings reveal the assimilatory sulfur metabolism in the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 and show how sulfur metabolism and oxidative stress response were remodeled and orchestrated to shape the biodesulfurization phenotype. Our findings not only explain the frequently encountered low catalytic activity of native fuel-biodesulfurizing bacteria but also uncover unprecedented potential targets in sulfur metabolism that could be exploited via metabolic engineering to boost the biodesulfurization catalytic activity, a prerequisite for commercial application.

Transcriptional Changes Involved in Atrophying Muscles during Prolonged Fasting in Rats.

Food deprivation resulting in muscle atrophy may be detrimental to health. To better understand how muscle mass is regulated during such a nutritional challenge, the current study deciphered muscle responses during phase 2 (P2, protein sparing) and phase 3 (P3, protein mobilization) of prolonged fasting in rats. This was done using transcriptomics analysis and a series of biochemistry measurements. The main findings highlight changes for plasma catabolic and anabolic stimuli, as well as for muscle transcriptome, energy metabolism, and oxidative stress. Changes were generally consistent with the intense use of lipids as fuels during P2. They also reflected increased muscle protein degradation and repressed synthesis, in a more marked manner during P3 than P2 compared to the fed state. Nevertheless, several unexpected changes appeared to be in favor of muscle protein synthesis during fasting, notably at the level of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, transcription and translation processes, and the response to oxidative stress. Such mechanisms might promote protein sparing during P2 and prepare the restoration of the protein compartment during P3 in anticipation of food intake for optimizing the effects of an upcoming refeeding, thereby promoting body maintenance and survival. Future studies should examine relevance of such targets for improving nitrogen balance during catabolic diseases.

Phylogenomic Classification and Biosynthetic Potential of the Fossil Fuel-Biodesulfurizing Rhodococcus Strain IGTS8.

Rhodococcus strain IGTS8 is the most extensively studied model bacterium for biodesulfurization of fossil fuels via the non-destructive sulfur-specific 4S pathway. This strain was initially assigned to Rhodococcus rhodochrous and later to Rhodococcus erythropolis thus making its taxonomic status debatable and reflecting the limited resolution of methods available at the time. In this study, phylogenomic analyses of the whole genome sequences of strain IGTS8 and closely related rhodococci showed that R. erythropolis and Rhodococcus qingshengii are very closely related species, that Rhodococcus strain IGTS8 is a R. qingshengii strain and that several strains identified as R. erythropolis should be re-classified as R. qingshengii. The genomes of strains assigned to these species contain potentially novel biosynthetic gene clusters showing that members of these taxa should be given greater importance in the search for new antimicrobials and other industrially important biomolecules. The plasmid-borne dsz operon encoding fossil fuel desulfurization enzymes was present in R. qingshengii IGTS8 and R. erythropolis XP suggesting that it might be transferable between members of these species.

Human proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin.

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.

Alterations in rat adipose tissue transcriptome and proteome in response to prolonged fasting.

Various pathophysiological situations of negative energy balance involve the intense depletion of the body's energy reserves. White adipose tissue is a central place to store energy and a major endocrine organ. As a model of choice to better understand how the white adipose tissue dynamically responds to changes in substrate availability, we used the prolonged fasting paradigm, which is characterized by successive periods of stimulated (phase 2) and then reduced (phase 3) lipid mobilization/utilization. Using omics analyses, we report a regulatory transcriptional program in rat epididymal (EPI) adipose tissue favoring lipolysis during phase 2 and repressing it during phase 3. Changes in gene expression levels of lipases, lipid droplet-associated factors, and the proteins involved in cAMP-dependent and cAMP-independent regulation of lipolysis are highlighted. The mRNA and circulating levels of adipose-secreted factors were consistent with the repression of insulin signaling during prolonged fasting. Other molecular responses are discussed, including the regulation of leptin and adiponectin levels, the specific changes reflecting an increased fibrinolysis and a possible protein catabolism-related energy saving mechanism in late fasting. Finally, some differences between internal and subcutaneous (SC) adipose tissues are also reported. These data provide a comprehensive molecular basis of adipose tissue responses when facing a major energetic challenge.

General lack of structural characterization of chemically synthesized long peptides.

Many peptide chemistry scientists have been reporting extremely interesting work on the basis of chemical peptides for which the only characterization was their purity, mass, and biological activity. It seems slightly overenthusiastic, as many of these structures should be thoroughly characterized first to demonstrate the uniqueness of the structure, as opposed to the uniqueness of the sequence. Among the peptides of identical sequences in the final chemical preparation, what amount of well-folded peptide supports the measured activity? The activity of a peptide preparation cannot prove the purity of the desired peptide. Therefore, greater care should be taken in characterizing peptides, particularly those coming from chemical synthesis. At a time when the pharmaceutical industry is changing its paradigm by moving substantially from small molecules to biologics to better serve patients' needs, it is important to understand the limitations of the descriptions of these products and to start to apply the same "good laboratory practices" to our peptide research. Here, we attempt to delineate how synthetic peptides are described and characterized and what will be needed to describe them in regards to how they are well-folded and homogeneous in their tertiary structure. Older studies were done when the tools were not yet discovered, but more recent publications are still lacking proper descriptions of these peptides. Modern tools of analysis are capable of segregating folded and unfolded peptides, even if the preparation is biologically active.

In-Depth Proteome Analysis Highlights HepaRG Cells as a Versatile Cell System Surrogate for Primary Human Hepatocytes.

Of the hepatic cell lines developed for in vitro studies of hepatic functions as alternatives to primary human hepatocytes, many have lost major liver-like functions, but not HepaRG cells. The increasing use of the latter worldwide raises the need for establishing the reference functional status of early biobanked HepaRG cells. Using deep proteome and secretome analyses, the levels of master regulators of the hepatic phenotype and of the structural elements ensuring biliary polarity were found to be close to those in primary hepatocytes. HepaRG cells proved to be highly differentiated, with functional mitochondria, hepatokine secretion abilities, and an adequate response to insulin. Among differences between primary human hepatocytes and HepaRG cells, the factors that possibly support HepaRG transdifferentiation properties are discussed. The HepaRG cell system thus appears as a robust surrogate for primary hepatocytes, which is versatile enough to study not only xenobiotic detoxification, but also the control of hepatic energy metabolism, secretory function and disease-related mechanisms.

An essential role for α4A-tubulin in platelet biogenesis.

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although ß1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to ß1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in Tuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

Stable isotope-labelled morphine to study in vivo central and peripheral morphine glucuronidation and brain transport in tolerant mice.

BACKGROUND AND PURPOSE: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels. EXPERIMENTAL APPROACH: Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms. KEY RESULTS: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study. CONCLUSIONS AND IMPLICATIONS: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine.

Multi-OMICS analyses unveil STAT1 as a potential modifier gene in mevalonate kinase deficiency.

OBJECTIVES: The objective of the present study was to explain why two siblings carrying both the same homozygous pathogenic mutation for the autoinflammatory disease hyper IgD syndrome, show opposite phenotypes, that is, the first being asymptomatic, the second presenting all classical characteristics of the disease. METHODS: Where single omics (mainly exome) analysis fails to identify culprit genes/mutations in human complex diseases, multiomics analyses may provide solutions, although this has been seldom used in a clinical setting. Here we combine exome, transcriptome and proteome analyses to decipher at a molecular level, the phenotypic differences between the two siblings. RESULTS: This multiomics approach led to the identification of a single gene-STAT1-which harboured a rare missense variant and showed a significant overexpression of both mRNA and protein in the symptomatic versus the asymptomatic sister. This variant was shown to be of gain of function nature, involved in an increased activation of the Janus kinase/signal transducer and activator of transcription signalling (JAK/STAT) pathway, known to play a critical role in inflammatory diseases and for which specific biotherapies presently exist. Pathway analyses based on information from differentially expressed transcripts and proteins confirmed the central role of STAT1 in the proposed regulatory network leading to an increased inflammatory phenotype in the symptomatic sibling. CONCLUSIONS: This study demonstrates the power of a multiomics approach to uncover potential clinically actionable targets for a personalised therapy. In more general terms, we provide a proteogenomics analysis pipeline that takes advantage of subject-specific genomic and transcriptomic information to improve protein identification and hence advance individualised medicine.

Extended investigation of tube-gel sample preparation: a versatile and simple choice for high throughput quantitative proteomics.

Sample preparation for quantitative proteomics is a crucial step to ensure the repeatability and the accuracy of the results. However, there is no universal method compatible with the wide variety of protein extraction buffers currently used. We have recently demonstrated the compatibility of tube-gel with SDS-based buffers and its efficiency for label-free quantitative proteomics by comparing it to stacking gel and liquid digestion. Here, we investigated the compatibility of tube-gel with alternatives to SDS-based buffers allowing notably the extraction of proteins in various pH conditions. We also explored the use of photopolymerization to extend the number of possibilities, as it is compatible with a wide range of pH and is non-oxidative. To achieve this goal, we compared six extraction buffers in combination with two polymerization conditions to further optimize the tube-gel protocol and evaluate its versatility. Identification and quantitative results demonstrated the compatibility of tube-gel with all tested conditions by overall raising quite comparable results. In conclusion, tube-gel is a versatile and simple sample preparation method for large-scale quantitative proteomics applications. Complete datasets are available via ProteomeXchange with identifier PXD008656.

The Circulating Protease Persephone Is an Immune Sensor for Microbial Proteolytic Activities Upstream of the Drosophila Toll Pathway.

Microbial or endogenous molecular patterns as well as pathogen functional features can activate innate immune systems. Whereas detection of infection by pattern recognition receptors has been investigated in details, sensing of virulence factors activities remains less characterized. In Drosophila, genetic evidences indicate that the serine protease Persephone belongs to a danger pathway activated by abnormal proteolytic activities to induce Toll signaling. However, neither the activation mechanism of this pathway nor its specificity has been determined. Here, we identify a unique region in the pro-domain of Persephone that functions as bait for exogenous proteases independently of their origin, type, or specificity. Cleavage in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous cysteine cathepsin 26-29-p. Our results establish Persephone itself as an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns.

Dual Data-Independent Acquisition Approach Combining Global HCP Profiling and Absolute Quantification of Key Impurities during Bioprocess Development.

Host cell proteins (HCP) are a major class of impurities derived from recombinant protein production processes. While HCP are usually monitored by ELISA, mass spectrometry (MS)-based approaches are emerging as promising orthogonal methods. Here, we developed an original method relying on data-independent acquisition (DIA) coupling global HCP amount estimation (Top 3) and absolute quantification with isotope dilution (ID). The method named Top 3-ID-DIA was benchmarked against ELISA and a gold-standard selected reaction monitoring assay (ID-SRM). Various samples generated at different steps and conditions of the purification process, including different culture durations, harvest procedures, and purification protocols were used to compare the methods. Overall, HCP were quantified over 5 orders of magnitude and down to the sub-ppm level. The Top 3-ID-DIA strategy proved to be equivalent to the gold-standard ID-SRM in terms of sensitivity (1-10 ppm), accuracy, and precision. Moreover, 81% of the Top 3 estimations were accurate within a factor of 2 when compared to ID-SRM. Thus, our approach aggregates global HCP profiling for comprehensive process understanding with absolute quantification of key HCP within a single analysis and provides an improved support for bioprocess development and product purity assessment.

Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts.

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 PDF specific activity to values similar to those of bacterial PDFs. We next show that Vp16 PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.

Developing a reference system for the IFCC standardization of HbA2.

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of ß-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

An evolutionary conserved zinc finger protein is involved in Toxoplasma gondii mRNA nuclear export.

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases affecting humans and livestock. There is an urgent need for new medicines that will target apicomplexan-specific pathways. We characterized a Toxoplasma gondii C2H2 zinc finger protein, named TgZNF2, which is conserved among eukaryotes. We constructed an inducible KO strain (iKO-TgZNF2) for this gene where the tgznf2 gene expression is repressed in the presence of a tetracycline analog (ATc). We showed that the iKO-TgZNF2 parasites are unable to proliferate after depletion of the TgZNF2 protein. Complementation with a full length copy of the gene restores the phenotype Moreover, the homolog of this protein in the related apicomplexan Plasmodium falciparum was shown to efficiently rescue the phenotype, suggesting that this pathway is likely conserved among apicomplexan parasites. We demonstrated that the iKO-mutant lacking TgZNF2 are arrested during the cell cycle during the G1 phase. We identified potential protein partners of this protein among which are spliceosomal complex and mRNA nuclear export components. We confirmed that TgZNF2 is able to bind in vivo to transcripts but splicing is not perturbed in the ATc-treated parasites. Instead, we demonstrated that TgZNF2 depletion leads to the sequestration of polyA+ mRNAs in the nucleus while ribosomal RNAs are not affected. We discovered a conserved protein with specific apicomplexan functional properties that is essential for the survival of T. gondii. TgZNF2 may be crucial to ensure the correct polyA+ mRNA nuclear export, a function that is conserved in P. falciparum.

A ruthenium anticancer compound interacts with histones and impacts differently on epigenetic and death pathways compared to cisplatin.

Ruthenium complexes are considered as potential replacements for platinum compounds in oncotherapy. Their clinical development is handicapped by a lack of consensus on their mode of action. In this study, we identify three histones (H3.1, H2A, H2B) as possible targets for an anticancer redox organoruthenium compound (RDC11). Using purified histones, we confirmed an interaction between the ruthenium complex and histones that impacted on histone complex formation. A comparative study of the ruthenium complex versus cisplatin showed differential epigenetic modifications on histone H3 that correlated with differential expression of histone deacetylase (HDAC) genes. We then characterized the impact of these epigenetic modifications on signaling pathways employing a transcriptomic approach. Clustering analyses showed gene expression signatures specific for cisplatin (42%) and for the ruthenium complex (30%). Signaling pathway analyses pointed to specificities distinguishing the ruthenium complex from cisplatin. For instance, cisplatin triggered preferentially p53 and folate biosynthesis while the ruthenium complex induced endoplasmic reticulum stress and trans-sulfuration pathways. To further understand the role of HDACs in these regulations, we used suberanilohydroxamic acid (SAHA) and showed that it synergized with cisplatin cytotoxicity while antagonizing the ruthenium complex activity. This study provides critical information for the characterization of signaling pathways differentiating both compounds, in particular, by the identification of a non-DNA direct target for an organoruthenium complex.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding.

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.