The Journey of Lipoproteins Through the Cell: One Birthplace, Multiple Destinations.

Bacterial lipoproteins are a very diverse group of proteins characterized by the presence of an N-terminal lipid moiety that serves as a membrane anchor. Lipoproteins have a wide variety of crucial functions, ranging from envelope biogenesis to stress response. In Gram-negative bacteria, lipoproteins can be targeted to various destinations in the cell, including the periplasmic side of the cytoplasmic or outer membrane, the cell surface or the external milieu. The sorting mechanisms have been studied in detail in Escherichia coli, but exceptions to the rules established in this model bacterium exist in other bacteria. In this chapter, we will present the current knowledge on lipoprotein sorting in the cell. Our particular focus will be on the surface-exposed lipoproteins that appear to be much more common than previously assumed. We will discuss the different targeting strategies, provide numerous examples of surface-exposed lipoproteins and discuss the techniques used to assess their surface exposure.

[A new protein system protects single cysteines against oxidative stress]. / Un nouveau système protéique protège les cystéines célibataires contre le stress oxydant.

The Escherichia coli periplasm contains several proteins from the thioredoxin family. DsbA and Dsbc interact with unfolded proteins to catalyze disulfide bond formation or isomerisation, respectively. The function of a third protein, DsbG, had remained elusive. By trapping DsbG attached to three of its substrates, we made the intriguing discovery that DsbG interacts with folded proteins possessing only one cysteine residue in their sequence. This residue is vulnerable to oxidation and forms a sulfenic acid in vitro. We sought to determine whether this cysteine is also sulfenylated in vivo, which led us to observe extensive sulfenic acid formation in the periplasm, especially in dsbcdsbG strains. Thus, by chasing the substrates of DsbG, we uncovered a new reducing system that is involved in sulfenic acid reduction on a global level (Depuydt et al., Science 326 (2009), 1109-1111). DsbG appears to be a key player in that system. Our work reveals one potentially widespread mechanism whereby the very reactive sulfenic acid modification can be controlled in the cellular environment.

[Unravelling the mechanisms of the biogenesis of the outer membrane of Gram-negative bacteria: a step toward the development of new antibiotics]. / Percer le mystere des mécanismes de biogenèse de la membrane externe des bactéries a Gram-négatif: une etape vers le développement de nouveaux antibiotiques.

The outer membrane of Gram negative bacteria such as Escherichia coli is a permeability barrier that is essential for the viability of Gram-negative bacteria and protects them against antimicrobial drugs, including hydrophobic antibiotics. Outer membrane components, including phospholipids, lipopolysaccharids and proteins are synthesized in the cytoplasm and the cytoplasmic membrane. The mechanisms by which unfolded proteins and lipids are then transported through the hydrophilic periplasm and are inserted in the outer membrane are essentially unknown. Our overall goal is to solve the fascinating problem of how such a complex macromolecular structure is assembled in a compartment devoid of obvious energy sources. Moreover, the proteins that are involved in OM biogenesis are also attractive targets for the design of new antibiotics and anti-inflammatory drugs. Developing new antibiotics active against E. coli and other Gram negative bacteria is criticial because the number of E. coli strains that are resistant to antibiotics is rapidly rising. We will describe results obtained recently in our laboratory that allowed us to characterize several periplasmic chaperones involved in the folding of envelope proteins.

Galectin-3 immunodetection in follicular thyroid neoplasms: a prospective study on fine-needle aspiration samples.

Fine-needle aspiration cytology, which is well established to be accurate for the diagnosis of thyroid cancer, may be inconclusive for the follicular thyroid neoplasms. As galectin-3 was suggested to be a marker of malignant thyrocytes, we investigated whether this protein might be helpful in the diagnosis of aspirates classified as undeterminate by cytology. After establishing an easy processing of aspirates for galectin-3 immunodetection, a series of aspirates categorised as benign (n=63), malignant (n=17) or undeterminate (n=34) was prospectively analysed for galectin-3. Only the patients with malignant or undeterminate lesions underwent surgery. Most lesions (86%) diagnosed as malignant by cytology or after surgery were positive for galectin-3. The majority of lesions (94%) classified as benign by cytology or after surgery was negative for galectin-3. The positive and negative predictive values were 83 and 95%, respectively. When focusing on the undeterminate lesions, the sensitivity and specificity were 75 and 90%, respectively, while the positive and negative predictive values were 82 and 87%, respectively. The specificity and the positive predictive value were higher (100%) when considering the percentage of stained cells. Altogether these results show that galectin-3 constitutes a useful marker in the diagnosis of thyroid lesions classified as undeterminate by conventional cytology.

Tissue distribution of the murine phosphomannomutases Pmm1 and Pmm2 during brain development.

The most common type of the congenital disorders of glycosylation, CDG-Ia, is caused by mutations in the human PMM2 gene, reducing phosphomannomutase (PMM) activity. The PMM2 mutations mainly lead to neurological symptoms, while other tissues are only variably affected. Another phosphomannomutase, PMM1, is present at high levels in the brain. This raises the question why PMM1 does not compensate for the reduced PMM2 activity during CDG-Ia pathogenesis. We compared the expression profile of the murine Pmm1 and Pmm2 mRNA and protein in prenatal and postnatal mouse brain at the histological level. We observed a considerable expression of both Pmms in different regions of the embryonic and adult mouse brain. Surprisingly, the expression patterns were largely overlapping. This data indicates that expression differences on the cellular and tissue level are an unlikely explanation for the absence of functional compensation. These results suggest that Pmm1 in vivo does not exert the phosphomannomutase-like activity seen in biochemical assays, but either acts on as yet unidentified specific substrates or fulfils entirely different functions.

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from Trypanosoma brucei: metal-ion dependency and phosphoenzyme formation.

Recombinant cofactor-independent phosphoglycerate mutase from Trypanosoma brucei was inactivated by EDTA, and reactivated by Co(2+) much more than by Mn(2+) or Fe(2+). It displayed a minor phosphoglycerate phosphatase activity, which was stimulated by Mn(2+) more than by Co(2+). Upon incubation with [(32)P]phosphoglycerate, radioactivity was incorporated into the enzyme, most particularly in the presence of Mn(2+) or Fe(2+). The phosphorylated residue was identified by tandem mass spectrometry as Ser74, a residue homologous to the phosphorylated serine in alkaline phosphatase. However, the rates of formation and of disappearance of this phosphoenzyme were quite low compared to the mutase reaction. This and other properties indicated that the observed phosphoenzyme is an intermediate in the minor phosphatase activity rather than in the phosphomutase reaction.

Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases.

Phosphoserine phosphatase belongs to a new class of phosphotransferases forming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases. The phosphorylated residue was identified as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with NaBH(4) or alkaline [(18)O]H(2)O. Incubation of native phosphoserine phosphatase with phosphoserine in [(18)O]H(2)O did not result in (18)O incorporation in residue Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-type ATPases, by attack of the phosphorus atom. Mutagenesis studies bearing on conserved residues indicated that four conservative changes either did not affect (S109T) or caused a moderate decrease in activity (G178A, D179E, and D183E). Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by >/=99% (D179N, D183N, K158A, and K158R). Mutations G178A and D179N decreased the affinity for phosphoserine, suggesting that these residues participate in the binding of the substrate. Mutations of Asp-179 decreased the affinity for Mg(2+), indicating that this residue interacts with the cation. Thus, investigated residues appear to play an important role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases.

Identification of the cDNA encoding human 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway(1).

We report the sequence of a human cDNA encoding a protein homologous to devB (a bacterial gene often found in proximity to the gene encoding glucose-6-phosphate dehydrogenase in bacterial genomes) and to the C-terminal part of human hexose-6-phosphate dehydrogenase. The protein was expressed in Escherichia coli, purified and shown to be 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway. Sequence analysis indicates that bacterial devB proteins, the C-terminal part of hexose-6-phosphate dehydrogenase and yeast Sol1-4 proteins are most likely also 6-phosphogluconolactonases and that these proteins are related to glucosamine-6-phosphate isomerases.

Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes.

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet. 19, 88-92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of approximately 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.

A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif.

When incubated with their substrates, human phosphomannomutase and L-3-phosphoserine phosphatase are known to form phosphoenzymes with chemical characteristics of an acyl-phosphate. The phosphorylated residue in phosphomannomutase has now been identified by mass spectrometry after reduction of the phosphoenzyme with tritiated borohydride and trypsin digestion. It is the first aspartate in a conserved DVDGT motif. Replacement of either aspartate of this motif by asparagine or glutamate resulted in complete inactivation of the enzyme. The same mutations performed in the DXDST motif of L-3-phosphoserine phosphatase also resulted in complete inactivation of the enzyme, except for the replacement of the second aspartate by glutamate, which reduced the activity by only about 40%. This suggests that the first aspartate of the motif is also the phosphorylated residue in L-3-phosphoserine phosphatase. Data banks contained seven other phosphomutases or phosphatases sharing a similar, totally conserved DXDX(T/V) motif at their amino terminus. One of these (beta-phosphoglucomutase) is shown to form a phosphoenzyme with the characteristics of an acyl-phosphate. In conclusion, phosphomannomutase and L-3-phosphoserine phosphatase belong to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor.

Phosphoserine phosphatase deficiency in a patient with Williams syndrome.

Decreased serine levels were found in plasma and cerebrospinal fluid (CSF) of a boy with pre- and postnatal growth retardation, moderate psychomotor retardation, and facial dysmorphism suggestive of Williams syndrome. Fluorescence in situ hybridisation with an elastin gene probe indicated the presence of a submicroscopic 7q11.23 deletion, confirming this diagnosis. Further investigation showed that the phosphoserine phosphatase (EC 3.1.3.3.) activity in lymphoblasts and fibroblasts amounted to about 25% of normal values. Oral serine normalised the plasma and CSF levels of this amino acid and seemed to have some clinical effect. These data suggest that the elastin gene and the phosphoserine phosphatase gene might be closely linked. This seems to be the first report of phosphoserine phosphatase deficiency.

Comparison of PMM1 with the phosphomannomutases expressed in rat liver and in human cells.

Carbohydrate-deficient glycoprotein syndrome type I (CDGI) is most often due to phosphomannomutase deficiency; paradoxically, the human phosphomannomutase gene PMM1 is located on chromosome 22, whereas the CDGI locus is on chromosome 16. We show that phosphomannomutases present in rat or human liver share with homogeneous recombinant PMM1 several kinetic properties and the ability to form an alkali- and NH2OH-sensitive phosphoenzyme with a subunit mass of approximately 30,000 Mr. However, they have a higher affinity for the activator mannose-1,6-bisphosphate than PMM1 and are not recognized by anti-PMM1 antibodies, indicating that they represent a related but different isozyme. Phosphomannomutases belong to a novel mutase family in which the active residue is a phosphoaspartyl or a phosphoglutamyl.

Human L-3-phosphoserine phosphatase: sequence, expression and evidence for a phosphoenzyme intermediate.

We report the sequence of the cDNA encoding human L-3-phosphoserine phosphatase. The encoded polypeptide contains 225 residues and shows 30% sequence identity with the Escherichia coli enzyme. The human protein was expressed in a bacterial expression system and purified. Similar to known L-3-phosphoserine phosphatases, it catalyzed the Mg2(+)-dependent hydrolysis of L-phosphoserine and an exchange reaction between L-serine and L-phosphoserine. In addition we found that the enzyme was phosphorylated upon incubation with L-[32P]phosphoserine, which indicates that the reaction mechanism proceeds via the formation of a phosphoryl-enzyme intermediate. The sensitivity of the phosphoryl-enzyme to alkali and to hydroxylamine suggests that an aspartyl- or a glutamyl-phosphate was formed. The nucleotide sequence of the cDNA described in this article has been deposited in the EMBL data base under accession number Y10275.

Sequence of a putative glucose 6-phosphate translocase, mutated in glycogen storage disease type Ib.

We report the sequence of a human cDNA that encodes a 46 kDa transmembrane protein homologous to bacterial transporters for phosphate esters. This protein presents at its carboxy terminus the consensus motif for retention in the endoplasmic reticulum. Northern blots of rat tissues indicate that the corresponding mRNA is mostly expressed in liver and kidney. In two patients with glycogen storage disease type Ib, mutations were observed that either replaced a conserved Gly to Cys or introduced a premature stop codon. The encoded protein is therefore most likely the glucose 6-phosphate translocase that is functionally associated with glucose-6-phosphatase.

Introduction of the cytological grading, the nuclear area, the DNA index and the DNA histogram type in the setting up of a score for ductal breast carcinoma.

The present study describes the setting up of a new score which makes it possible objectively to grade ductal breast carcinomas, i.e. not-otherwise-specified (NOS) cancers, on cellular material from fine-needle aspirations (FNAs). For this purpose FNAs from 252 patients--with NOS breast cancers--were smeared onto histological slides, fixed in an ethanol-formolacetic acid mixture, Feulgen-stained and analysed by means of a cell image processor. Four parameters were taken into account in setting up the score, namely the cytological prognostic grade (CPM) of malignancies similar to the Scarff-Bloom-Richardson (SBR) grading, the nuclear area (NA), the DNA index (DI) and the DNA histogram type (DHT). Each of these four parameters was considered as a "sub-score" which may take three values, i.e. 1, 2 and 3. The final result may thus range from 4 to 12. Subscores of 4 and 5 correspond to a cytological score of I, subscores of 6, 7 and 8 to a cytological score of II, sub-scores of 9 and 10 to a cytological score of III, and sub-scores of 11 and 12 to a cytological score of IV. In the present study, the results show 17% of CPM grade 1.52% of CPM grade II and 31% of CPM grade III cancers. All the cases exhibiting a cytological score of IV (5%) fully fit in with the CPM grade III cancers. In the same way, none of the cases exhibiting a score of I fit in with CPM grade III cancers. The cancers with a CPM grade II fit in with the scores of II and III. It thus seems possible to convert a three-value malignancy grading system (CPM and/or SBR grading) into a four-value one (cytological score). The main advantage in this latter type of system is that it becomes possible to split up the over-large group of CPM grade II cancers. As things stand, we are unable to give any prognostic value for the score proposed here because our study is prospective only. A study of this type has been necessary so as to provide against problems connected with ways of preserving specimens that might be used in a retrospective study. The bank of clinical and biological data now in existence must be allowed to mature for a number of years before the prognostic worth of the cytological score can be established, always assuming that such a value exists.