Targeted mutation and inactivation of the kinesin light chain 3 protein-encoding gene have no impact on mouse fertility†.

The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment.

Mutation of the MYH3 gene causes recessive cleft palate in Limousine cattle.

BACKGROUND: The palate is a structure separating the oral and nasal cavities and its integrity is essential for feeding and breathing. The total or partial opening of the palate is called a cleft palate and is a common malformation in mammals with environmental or hereditary aetiologies. Generally, it compromises life expectancy in the absence of surgical repair. A new form of non-syndromic cleft palate arose recently in Limousine cattle, with animals referred to the French National Observatory of Bovine Abnormalities since 2012. Since the number of affected animals has increased steadily, this study was undertaken to identify the cause of this disease. RESULTS: Based on pedigree analysis, occurrence of cleft palate in Limousine cattle was concordant with an autosomal recessive mode of inheritance. Genotyping of 16 affected animals and homozygosity mapping led to the identification of a single disease-associated haplotype on Bos taurus chromosome (BTA)19. The genome of two affected animals was sequenced, and their sequences were compared to the ARS-UCD1.2 reference genome to identify variants. The likely causal variants were compared to the variant database of the 1000 bull genome project and two fully linked mutations in exon 24 of the MYH3 (myosin heavy chain) gene were detected: a 1-bp non-synonymous substitution (BTA19:g.29609623A>G) and a 11-bp frameshift deletion (BTA19:g.29609605-29609615del). These two mutations were specific to the Limousine breed, with an estimated allele frequency of 2.4% and are predicted to be deleterious. The frameshift leads to a premature termination codon. Accordingly, mRNA and protein analyses in muscles from wild-type and affected animals revealed a decrease in MYH3 expression in affected animals, probably due to mRNA decay, as well as an absence of the MYH3 protein in these animals. MYH3 is mostly expressed in muscles, including craniofacial muscles, during embryogenesis, and its absence may impair palate formation. CONCLUSIONS: We describe a new form of hereditary cleft palate in Limousine cattle. We identified two fully linked and deleterious mutations, ultimately leading to the loss-of-function of the MYH3 protein. The mutations were included on the Illumina EuroG10k v8 and EuroGMD v1 SNP chips and are used to set up a reliable eradication strategy in the French Limousine breed.

Co-invalidation of Prnp and Sprn in FVB/N mice affects reproductive performances and highlight complex biological relationship between PrP and Shadoo.

Shadoo and PrP belongs to the same protein family, whose biological function remains poorly understood. Previous experiments reported potential functional redundancies or antagonisms between these two proteins, depending on the tissue analysed. While knockdown experiments suggested the requirement of Shadoo in the absence of PrP during early mouse embryogenesis, knockout ones, on the contrary, highlighted little impact, if any, of the double-knockout of these two loci. In the present study, we reinvestigated the phenotype associated with the concomitant knockout of these two genes using newly produced FVB/N Sprn knockout mice. In this genetic background, the combined two genes' knockout induces intra-uterine growth retardations, likely resulting from placental failures highlighted by transcriptomic analyses that revealed potential redundant or antagonist roles of these two proteins in different developmental-related pathways. It also induced an increased perinatal-lethality and ascertained the role of these two loci in the lactation process.

CEP250 is Required for Maintaining Centrosome Cohesion in the Germline and Fertility in Male Mice.

Male gametogenesis involves both mitotic divisions to amplify germ cell progenitors that gradually differentiate and meiotic divisions. Centrosomal regulation is essential for both types of divisions, with centrioles remaining tightly paired during the interphase. Here, we generated and characterized the phenotype of mutant mice devoid of Cep250/C-Nap1, a gene encoding for a docking protein for fibers linking centrioles, and characterized their phenotype. The Cep250 -/- mice presented with no major defects, apart from male infertility due to a reduction in the spermatogonial pool and the meiotic blockade. Spermatogonial stem cells expressing Zbtb16 were not affected, whereas the differentiating spermatogonia were vastly lost. These cells displayed abnormal γH2AX-staining, accompanied by an increase in the apoptotic rate. The few germ cells that survived at this stage, entered the meiotic prophase I and were arrested at a pachytene-like stage, likely due to synapsis defects and the unrepaired DNA double-strand breaks. In these cells, centrosomes split up precociously, with γ-tubulin foci being separated whereas these were closely associated in wild-type cells. Interestingly, this lack of cohesion was also observed in wild-type female meiocytes, likely explaining the normal fertility of Cep250 -/- female mice. Taken together, this study proposes a specific requirement of centrosome cohesion in the male germline, with a crucial role of CEP250 in both differentiating spermatogonia and meiotic spermatocytes.

The Prion-like protein Shadoo is involved in mouse embryonic and mammary development and differentiation.

Shadoo belongs to the prion protein family, an evolutionary conserved and extensively studied family due to the implication of PrP in Transmissible Spongiform Encephalopathies. However, the biological function of these genes remains poorly understood. While Sprn-knockdown experiments suggested an involvement of Shadoo during mouse embryonic development, Sprn-knockout experiments in 129Pas/C57BL/6J or 129Pas/FVB/NCr mice did not confirm it. In the present study, we analyzed the impact of Sprn gene invalidation in a pure FVB/NJ genetic background, using a zinc finger nuclease approach. The in-depth analysis of the derived knockout transgenic mice revealed a significant increase in embryonic lethality at early post-implantation stages, a growth retardation of young Sprn-knockout pups fed by wild type mice and a lactation defect of Sprn-knockout females. Histological and transcriptional analyses of knockout E7.5 embryos, E14.5 placentas and G7.5 mammary glands revealed specific roles of the Shadoo protein in mouse early embryogenesis, tissue development and differentiation with a potential antagonist action between PrP and Shadoo. This study thus highlights the entanglement between the proteins of the prion family.

DNAJC2 is required for mouse early embryonic development.

DNAJC2 protein, also known as ZRF1 or MPP11, acts both as chaperone and as chromatin regulator. It is involved in stem cell differentiation and its expression is associated with various cancer malignancies. However, the role of Dnajc2 gene during mouse embryogenesis has not been assessed so far. To this aim, we invalidated Dnajc2 gene in FVB/Nj mice using the CrispR/Cas9 approach. We showed that this invalidation leads to the early post-implantation lethality of the nullizygous embryos. Furthermore, using siRNAs against Dnajc2 in mouse 1-cell embryos, we showed that maternal Dnajc2 mRNAs may allow for the early preimplantation development of these embryos. Altogether, these data demonstrate for the first time the requirement of DNAJC2 for early mouse embryogenesis.

Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Bovine and murine models highlight novel roles for SLC25A46 in mitochondrial dynamics and metabolism, with implications for human and animal health.

Neuropathies are neurodegenerative diseases affecting humans and other mammals. Many genetic causes have been identified so far, including mutations of genes encoding proteins involved in mitochondrial dynamics. Recently, the "Turning calves syndrome", a novel sensorimotor polyneuropathy was described in the French Rouge-des-Prés cattle breed. In the present study, we determined that this hereditary disease resulted from a single nucleotide substitution in SLC25A46, a gene encoding a protein of the mitochondrial carrier family. This mutation caused an apparent damaging amino-acid substitution. To better understand the function of this protein, we knocked out the Slc25a46 gene in a mouse model. This alteration affected not only the nervous system but also altered general metabolism, resulting in premature mortality. Based on optic microscopy examination, electron microscopy and on biochemical, metabolic and proteomic analyses, we showed that the Slc25a46 disruption caused a fusion/fission imbalance and an abnormal mitochondrial architecture that disturbed mitochondrial metabolism. These data extended the range of phenotypes associated with Slc25a46 dysfunction. Moreover, this Slc25a46 knock-out mouse model should be useful to further elucidate the role of SLC25A46 in mitochondrial dynamics.

Sialylated Fetuin-A as a candidate predictive biomarker for successful grass pollen allergen immunotherapy.

BACKGROUND: Eligibility to immunotherapy is based on the determination of IgE reactivity to a specific allergen by means of skin prick or in vitro testing. Biomarkers predicting the likelihood of clinical improvement during immunotherapy would significantly improve patient selection. METHODS: Proteins were differentially assessed by using 2-dimensional differential gel electrophoresis and label-free mass spectrometry in pretreatment sera obtained from clinical responders and nonresponders within a cohort of 82 patients with grass pollen allergy receiving sublingual immunotherapy or placebo. Functional studies of Fetuin-A (FetA) were conducted by using gene silencing in a mouse asthma model, human dendritic cell in vitro stimulation assays, and surface plasmon resonance. RESULTS: Analysis by using quantitative proteomics of pretreatment sera from patients with grass pollen allergy reveals that high levels of O-glycosylated sialylated FetA isoforms are found in patients exhibiting a strong decrease in rhinoconjunctivitis symptoms after sublingual immunotherapy. Although FetA is involved in numerous inflammatory conditions, its potential role in allergy is unknown. In vivo silencing of the FETUA gene in BALB/c mice results in a dramatic upregulation of airway hyperresponsiveness, lung resistance, and TH2 responses after allergic sensitization to ovalbumin. Both sialylated and nonsialytated FetA bind to LPS, but only the former synergizes with LPS and grass pollen or mite allergens to enhance the Toll-like receptor 4-mediated proallergic properties of human dendritic cells. CONCLUSIONS: As a reflection of the patient's inflammatory status, pretreatment levels of sialylated FetA in the blood are indicative of the likelihood of clinical responses during grass pollen immunotherapy.

Bacterial protein signals are associated with Crohn's disease.

OBJECTIVE: No Crohn's disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. DESIGN: We first developed and validated a workflow-including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS-to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. RESULTS: Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. CONCLUSIONS: This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis.

Evolution of major milk proteins in Mus musculus and Mus spretus mouse species: a genoproteomic analysis.

BACKGROUND: Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of Mus spretus and Mus musculus species, respectively. RESULTS: The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L vs. 125 ± 12 g/L, respectively). Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (ß and αs1) and the whey acidic protein (WAP), showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas Csn1s1 (11), Csn2 (7) and Wap (8) genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas Wap gene. CONCLUSION: SNP frequencies found in three milk protein-encoding genes between Mus spretus and Mus musculus is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence), already identified in casein genes from other species, likely explain the existence of multiple αs1-casein isoforms both in SEG/Pas and C57BL/6J strains. Finally, we propose a possible mechanism by which the hallmark tandem duplication of a 18-nt exon (14 copies) may have occurred in the mouse genome.

An iterative workflow for mining the human intestinal metaproteome.

BACKGROUND: Peptide spectrum matching (PSM) is the standard method in shotgun proteomics data analysis. It relies on the availability of an accurate and complete sample proteome that is used to make interpretation of the spectra feasible. Although this procedure has proven to be effective in many proteomics studies, the approach has limitations when applied on complex samples of microbial communities, such as those found in the human intestinal tract. Metagenome studies have indicated that the human intestinal microbiome contains over 100 times more genes than the human genome and it has been estimated that this ecosystem contains over 5000 bacterial species. The genomes of the vast majority of these species have not yet been sequenced and hence their proteomes remain unknown. To enable data analysis of shotgun proteomics data using PSM, and circumvent the lack of a defined matched metaproteome, an iterative workflow was developed that is based on a synthetic metaproteome and the developing metagenomic databases that are both representative for but not necessarily originating from the sample of interest. RESULTS: Two human fecal samples for which metagenomic data had been collected, were analyzed for their metaproteome using liquid chromatography-mass spectrometry and used to benchmark the developed iterative workflow to other methods. The results show that the developed method is able to detect over 3,000 peptides per fecal sample from the spectral data by circumventing the lack of a defined proteome without naive translation of matched metagenomes and cross-species peptide identification. CONCLUSIONS: The developed iterative workflow achieved an approximate two-fold increase in the amount of identified spectra at a false discovery rate of 1% and can be applied in metaproteomic studies of the human intestinal tract or other complex ecosystems.

Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry.

The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNA(Ile), the 2',3'-dialdehyde derivative of tRNA(Ile) was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNA(Ile) aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNA(Ile)ox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNA(Ile) and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence (601)KMSKS(605). Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [(32)P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.

Disulphide bonds in casein micelle from milk.

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.