Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase.

International audienceThe enzyme apolipoprotein N-acyltransferase (Lnt) is an integral membrane protein that catalyzes the last step in the post-translational modification of bacterial lipoproteins. Lnt undergoes covalent modification in the presence of phospholipids resulting in a thioester acyl-enzyme intermediate. It then transfers the acyl chain to the α-amino group of the N-terminal diacylglyceryl-modified cysteine of apolipoprotein, leading to the formation of mature triacylated lipoprotein. To gain insight into the catalytic mechanism of this two-step reaction, we overproduced and purified the enzyme of Escherichia coli and studied its N-acyltransferase activity using a novel in vitro assay. The purified enzyme was fully active, as judged by its ability to form a stable thioester acyl-enzyme intermediate and N-acylate the apo-form of the murein lipoprotein Lpp in vitro. Incorporation of [(3)H]palmitate and mass spectrometry analysis demonstrated that Lnt recognized the synthetic diacylglyceryl-modified lipopeptide FSL-1 as a substrate in a mixed micelle assay. Kinetics of Lnt using phosphatidylethanolamine as an acyl donor and FSL-1 as a substrate were consistent with a ping-pong type mechanism, demonstrating slow acyl-enzyme intermediate formation and rapid N-acyl transfer to the apolipopeptide in vitro. In contrast to earlier in vitro observations, the N-acyltransferase activity was strongly affected by the phospholipid headgroup and acyl chain composition

Total ankle replacement in young patients

High physical demand and younger age are currently considered contraindications for total ankle replacement. The number of Total Ankle Replacements (TAR) is widespread increasing and indications are expanding thanks to a steady improvement in prosthetic designs and better outcome. Commentary of the literature: in 1999 a study of 100 uncemented STAR\u2122 (Waldemar-Link, Hamburg, Germany) prostheses showed a survival rate of 75% at 6.8 years in patients under 50 years old. Other studies (es, Barg et Al.) shows the risk of failure age-related in young patients compared to older group. A report of 780 TAR from the Swedish Ankle Register showed patients with primary or post-traumatic osteoarthritis under  60 years of age to have a 1.8 higher chance of revision compared to older patients. Discussion: ankle replacement has been traditionally reserved for patients older 50 years old and with low physical demand. Contrariwise this belief, TAR have already been used with a wide range of ages, sometimes even patients younger than 30 years old. Most of the "negative" score and results showed before are related to "second-generation" prosthetic designs, while recent studies used a "third-generation" prosthetic design. Conclusions: recent evidences showed better clinical results and higher satisfaction in people under the age of 50 compared to ankle arthrodesis with comparable rate of complications and survivorship. Younger people will have however a higher rate of reoperation but in the meantime, they will prevent progressive degeneration of adjacent joints

Navicular tenosuspension with anterior tibialis tendon (Young procedure) associated to calcaneo-stop for the treatment of paediatric flexible flatfoot: clinical and ultrasound study

Background and aim of the work: Flexible flatfoot is one of the most common deformities in pediatric orthopaedics. Arthroeresis procedures are designed to correct this deformity. Among them, calcaneostop is a procedure with both biomechanical and proprioceptive properties. There could be other surgical procedure combined, such as a percutaneous Achilles tendon lengthening and the Gould tibialis posterior retension or Young tibialis anterior navicular tenosuspension. This study analyzed the clinical and sonographic results of 36 patients following flexible flatfoot surgical treatment with a calcaneo-stop arthroeresis combined with Achilles lengthening and a Young procedure. Methods: From March 2001 to August 2014, 36 patients (54 feet) were treated with calcaneo-stop arthroeresis, percutaneous Achilles tendon lengthening and Young\u2019s tenosuspension. The clinical assessment and a sonography of the anterior tibialis tendon (ATT) were performed in all patients. Results: The average follow-up was 7.4 years (range 8 months-14 years) with a satisfactory outcome in 51 feet (94.5%). No major and minor complications were observed. In four cases the calcaneo-stop was removed for pain and low tolerance of the patient. The AOFAS score and the talocalcaneal angle did not have statistically significant in case of ATT was or not still inserted in the navicular at the follow-up. Conclusions: The calcaneo-stop procedure is a simple, reliable and minimally invasive procedure for the treatment of pediatric flexible flatfoot. Although the indications for the Young tenosuspension as an isolated procedure is very narrow, it can still be an effective procedure when combined to calcaneo-stop. The key to appropriate utilization is a thorough understanding of the biomechanics of the foot function and a specific appreciation of the function of the ATT

Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in; Corynebacterium glutamicum;

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys; +1; residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys; +1; , which can eventually be further modified. Here, we identified the; lgt; and; lspA; genes from; Corynebacterium glutamicum; and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a; C. glutamicum; maltose-binding lipoprotein, and LppX, a; Mycobacterium tuberculosis; lipoprotein. However, Lgt is not required for these proteins' signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in; C. glutamicum; the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification

Identification of a Small TAF Complex and Its Role in the Assembly of TAF-Containing Complexes

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation

The ppm Operon Is Essential for Acylation and Glycosylation of Lipoproteins in Corynebacterium glutamicum

BACKGROUND: Due to their contribution to bacterial virulence, lipoproteins and members of the lipoprotein biogenesis pathway represent potent drug targets. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), cleaved off their signal peptides by lipoprotein signal peptidase (Lsp) and, in Gram-negative bacteria, further triacylated by lipoprotein N-acyl transferase (Lnt). The existence of an active apolipoprotein N-acyltransferase (Ms-Ppm2) involved in the N-acylation of LppX was recently reported in M. smegmatis. Ms-Ppm2 is part of the ppm operon in which Ppm1, a polyprenol-monophosphomannose synthase, has been shown to be essential in lipoglycans synthesis but whose function in lipoprotein biosynthesis is completely unknown. RESULTS: In order to clarify the role of the ppm operon in lipoprotein biosynthesis, we investigated the post-translational modifications of two model lipoproteins (AmyE and LppX) in C. glutamicum Δppm1 and Δppm2 mutants. Our results show that both proteins are anchored into the membrane and that their N-termini are N-acylated by Cg-Ppm2. The acylated N-terminal peptide of LppX was also found to be modified by hexose moieties. This O-glycosylation is localized in the N-terminal peptide of LppX and disappeared in the Δppm1 mutant. While compromised in the absence of Cg-Ppm2, LppX O-glycosylation could be restored when Cg-Ppm1, Cg-Ppm2 or the homologous Mt-Ppm1 of M. tuberculosis was overexpressed. CONCLUSION: Together, these results show for the first time that Cg-Ppm1 (Ppm synthase) and Cg-Ppm2 (Lnt) operate in a common biosynthetic pathway in which lipoprotein N-acylation and glycosylation are tightly coupled

The in vivo mitochondrial two-step maturation of human frataxin.

International audienceDeficiency in the nuclear-encoded mitochondrial protein frataxin causes Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associating spinocerebellar ataxia and cardiomyopathy. Although the exact function of frataxin is still a matter of debate, it is widely accepted that frataxin is a mitochondrial iron chaperone involved in iron-sulfur cluster and heme biosynthesis. Frataxin is synthesized as a precursor polypeptide, directed to the mitochondrial matrix where it is proteolytically cleaved by the mitochondrial processing peptidase to the mature form via a processing intermediate. The mature form was initially reported to be encoded by amino acids 56-210 (m(56)-FXN). However, two independent reports have challenged these studies describing two different forms encoded by amino acids 78-210 (m(78)-FXN) and 81-210 (m(81)-FXN). Here, we provide evidence that mature human frataxin corresponds to m(81)-FXN, and can rescue the lethal phenotype of fibroblasts completely deleted for frataxin. Furthermore, our data demonstrate that the migration profile of frataxin depends on the experimental conditions, a behavior which most likely contributed to the confusion concerning the endogenous mature frataxin. Interestingly, we show that m(56)-FXN and m(78)-FXN can be generated when the normal maturation process of frataxin is impaired, although the physiological relevance is not clear. Furthermore, we determine that the d-FXN form, previously reported to be a degradation product, corresponds to m(78)-FXN. Finally, we demonstrate that all frataxin isoforms are generated and localized within the mitochondria. The clear identification of the N-terminus of mature FXN is an important step for designing therapeutic approaches for FRDA based on frataxin replacement

FAD/folate-dependent tRNA methyltransferase: flavin as a new methyl-transfer agent.

International audienceRNAs contain structurally and functionally important modified nucleosides. Methylation, the most frequent RNA modification in all living organisms, mostly relies on SAM (S-adenosylmethionine)-dependent methyltransferases. TrmFO was recently discovered as a unique tRNA methyltransferase using instead methylenetetrahydrofolate and reduced flavin adenine dinucleotide (FAD) as essential cofactors, but its mechanism has remained elusive. Here, we report that TrmFO carries an active tRNA-methylating agent and characterize it as an original enzyme-methylene-FAD covalent adduct by mass spectrometry and a combination of spectroscopic and biochemical methods. Our data support a novel tRNA methylating mechanism