Production of a chiral alcohol, 1-(3,4-dihydroxyphenyl) ethanol, by mushroom tyrosinase

1-(3,4-Dihydroxyphenyl) ethanol was produced biocatalytically for the first time using mushroom tyrosinase. 4-Ethylphenol at 1 mM was consumed over 12 min giving 0.23 mM 4-ethylcatechol and 0.36 mM (R/S)-1-(3,4-dihydroxyphenyl) ethanol (ee 0.5 %). Mushroom tyrosinase consumed 4-ethylphenol at 6.7 μmol min^?1 mg protein^?1 while the rates of formation of 4-ethylcatechol and 1-(3,4-dihydroxyphenyl) ethanol were 1.1 and 1.9 μmol min^?1 mg protein^?1. Addition of the ascorbic acid, as a reducing agent to biotransformation reactions, increased 4-ethylcatechol formation by 340 %. However, accumulation of 1-(3,4-dihydroxyphenyl) ethanol was not observed in the presence of ascorbic acid. While the 1-(3,4-dihydroxyphenyl) ethanol was racemic, it is the first chiral product produced by tyrosinase starting from a non-chiral substrate.     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

Deficient tryptophan catabolism along the kynurenine pathway reveals that the epididymis is in a unique tolerogenic state

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1(-/-)-deficient mice. In the caput epididymis of Ido1(-/-) animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.     

C2orf62 and TTC17 Are Involved in Actin Organization and Ciliogenesis in Zebrafish and Human

Vertebrate genomes contain around 20,000 protein-encoding genes, of which a large fraction is still not associated with specific functions. A major task in future genomics will thus be to assign physiological roles to all open reading frames revealed by genome sequencing. Here we show that C2orf62, a highly conserved protein with little homology to characterized proteins, is strongly expressed in testis in zebrafish and mammals, and in various types of ciliated cells during zebrafish development. By yeast two hybrid and GST pull-down, C2orf62 was shown to interact with TTC17, another uncharacterized protein. Depletion of either C2orf62 or TTC17 in human ciliated cells interferes with actin polymerization and reduces the number of primary cilia without changing their length. Zebrafish embryos injected with morpholinos against C2orf62 or TTC17, or with mRNA coding for the C2orf62 C-terminal part containing a RII dimerization/docking (R2D2) – like domain show morphological defects consistent with imperfect ciliogenesis. We provide here the first evidence for a C2orf62-TTC17 axis that would regulate actin polymerization and ciliogenesis.     

Field Trial Evaluation of the Performances of Point-of-Care Tests for Screening G6PD Deficiency in Cambodia

Background

User-friendly, accurate, point-of-care rapid tests to detect glucose-6-phosphate dehydrogenase deficiency (G6PDd) are urgently needed at peripheral level to safely recommend primaquine for malaria elimination.

Methods

The CareStart G6PD RDT (AccessBio, New Jersey, USA), a novel rapid diagnostic test and the most commonly used test, the fluorescent spot test (FST) were assessed against the quantitatively measured G6PD enzyme activity for detecting G6PDd. Subjects were healthy males and non-pregnant females aged 18 years or older residing in six villages in Pailin Province, western Cambodia.

Findings

Of the 938 subjects recruited, 74 (7.9%) were severe and moderately severe G6PD deficient (enzyme activity <30%), mostly in male population; population median G6PD activity was 12.0 UI/g Hb. The performances of the CareStart G6PD RDT and the FST, according to different cut-off values used to define G6PDd were very similar. For the detection of severe and moderately severe G6PDd (enzyme activity <30%, <3.6 UI/g Hb) in males and females, sensitivity and negative (normal status) predictive value were 100% for both point-of-care tools. When the G6PDd cut-off value increased (from <40% to <60%), the sensitivity for both PoCs decreased: 93.3% to 71.7% (CareStart G6PD RDT, p = 10⁻⁶) and 95.5% to 73.2% (FST, p = 10⁻⁶) while the specificity for both PoCs remained similar: 97.4% to 98.3% (CareStart G6PD RDT, p = 0.23) and 98.7% to 99.6% (FST, p = 0.06). The cut-off values for classifying individuals as normal were 4.0 UI/g Hb and 4.3 UI/g Hb for the CareStart G6PD RDT and the FST, respectively.

Conclusions

The CareStart G6PD RDT reliably detected moderate and severe G6PD deficient individuals (enzyme activity <30%), suggesting that this novel point-of-care is a promising tool for tailoring appropriate primaquine treatment for malaria elimination by excluding individuals with severe G6PDd for primaquine treatment.     

A new method of purification of proteasome substrates reveals polyubiquitination of 20 S proteasome subunits

The 26 S proteasome is implicated in the control of many major biological functions but a reliable method for the identification of its major substrates, i.e. polyubiquitin (Ub) conjugates, is still lacking. Based on the steps present in cells, i.e. recognition and deubiquitination, we developed an affinity matrix-based purification of polyUb conjugates suitable for any biological sample. Ub-conjugates were first purified from proteasome inhibitor-treated C2C12 cells using the Ub binding domains of the S5a proteasome subunit bound to an affinity matrix and then deubiquitinated by the catalytic domain of the USP2 enzyme. This two step purification of proteasome substrates involving both protein-protein interactions and enzyme-mediated release allowed highly specific isolation of polyUb 26 S proteasome substrates, which were then resolved on two-dimensional gels post-deubiquitination. To establish our method, we focused on a gel area where spots were best resolved. Surprisingly, spot analysis by mass spectrometry identified alpha2, alpha6, alpha7, beta2, beta3, beta4, and beta5 20 S proteasome subunits as potential substrates. Western blots using an anti-beta3 proteasome subunit antibody confirmed that high molecular weight forms of beta3 were present, particularly in proteasome inhibitor-treated cells. Sucrose gradients of cell lysates suggested that the proteasome was first disassembled before subunits were polyubiquitinated. Altogether, we provide a technique that enables large scale identification of 26 S proteasome substrates that should contribute to a better understanding of this proteolytic machinery in any living cell and/or organ/tissue. Furthermore, the data suggest that proteasome homeostasis involves an autoregulatory mechanism.     

C11orf83, a Mitochondrial Cardiolipin-Binding Protein Involved in bc1 Complex Assembly and Supercomplex Stabilization

Mammalian mitochondria may contain up to 1,500 different proteins, and many of them have neither been confidently identified nor characterized. In this study, we demonstrated that C11orf83, which was lacking experimental characterization, is a mitochondrial inner membrane protein facing the intermembrane space. This protein is specifically associated with the bc₁ complex of the electron transport chain and involved in the early stages of its assembly by stabilizing the bc₁ core complex. C11orf83 displays some overlapping functions with Cbp4p, a yeast bc₁ complex assembly factor. Therefore, we suggest that C11orf83, now called UQCC3, is the functional human equivalent of Cbp4p. In addition, C11orf83 depletion in HeLa cells caused abnormal crista morphology, higher sensitivity to apoptosis, a decreased ATP level due to impaired respiration and subtle, but significant, changes in cardiolipin composition. We showed that C11orf83 binds to cardiolipin by its α-helices 2 and 3 and is involved in the stabilization of bc₁ complex-containing supercomplexes, especially the III₂/IV supercomplex. We also demonstrated that the OMA1 metalloprotease cleaves C11orf83 in response to mitochondrial depolarization, suggesting a role in the selection of cells with damaged mitochondria for their subsequent elimination by apoptosis, as previously described for OPA1.     

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans–Brassica napus pathosystem

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.