Secretome analysis of chondroitin sulfate-treated chondrocytes reveals anti-angiogenic,anti-inflammatory and anti-catabolic properties

Introduction

Chondroitin sulfate (CS) is a symptomatic slow-acting drug for osteoarthritis (OA) widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli (IL-1β) is regulated by CS, employing a novel quantitative proteomic approach.

Methods

Human articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1β 5 ng/ml with or without CS pretreatment (200 µg/ml). Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results.

Results

We could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance (six increased and 12 decreased). In normal chondrocytes stimulated with IL-1β, CS reduces inflammation directly by decreasing the presence of several complement components (CFAB, C1S, CO3, and C1R) and also indirectly by increasing proteins such as TNFα-induced protein (TSG6). TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation (observed in MMP1 and MMP3 levels). Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1.

Conclusion

We have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1β. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.     

Multiple transisthmian divergences,extensive cryptic diversity,occasional long‐distance dispersal,and biogeographic patterns in a marine coastal isopod with an amphi‐American distribution

Excirolana braziliensis is a coastal intertidal isopod with a broad distribution spanning the Atlantic and Pacific tropical and temperate coasts of the American continent. Two separate regional studies (one in Panama and one in Chile) revealed the presence of highly genetically divergent lineages, implying that this taxon constitutes a cryptic species complex. The relationships among the lineages found in these two different regions and in the rest of the distribution, however, remain unknown. To better understand the phylogeographic patterns of E. braziliensis, we conducted phylogenetic analyses of specimens from much of its entire range. We obtained DNA sequences for fragments of four mitochondrial genes (16S rDNA, 12S rDNA, COI, and Cytb) and also used publicly available sequences. We conducted maximum likelihood and Bayesian phylogenetic reconstruction methods. Phylogeographic patterns revealed the following: (1) new highly divergent lineages of E. braziliensis; (2) three instances of Atlantic–Pacific divergences, some of which appear to predate the closure of the Isthmus of Panama; (3) the distributional limit of highly divergent lineages found in Brazil coincides with the boundary between two major marine coastal provinces; (4) evidence of recent long‐distance dispersal in the Caribbean; and (5) populations in the Gulf of California have closer affinities with lineages further south in the Pacific, which contrasts with the closer affinity with the Caribbean reported for other intertidal organisms. The high levels of cryptic diversity detected also bring about challenges for the conservation of this isopod and its fragile environment, the sandy shores. Our findings underscore the importance of comprehensive geographic sampling for phylogeographic and taxonomical studies of broadly distributed putative species harboring extensive cryptic diversity.     

Maturation of the mammalian secretome

A recent use of quantitative proteomics to determine the constituents of the endoplasmic reticulum and Golgi complex is discussed in the light of other available methodologies for cataloging the proteins associated with the mammalian secretory pathway.     

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.     

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.Arsenic is a carcinogen that ranks first on the Superfund List of Hazardous Substances (www.atsdr.cdc.gov). As a consequence of its environmental ubiquity, nearly every organism, from bacteria to humans, has genes that confer resistance to arsenic (1). The most common mechanism of arsenite resistance is efflux from cells catalyzed by members of three unrelated families of transporters. Homologues of the Mrp members of the ATP-binding cassette superfamily catalyze ATP-dependent pumping of As(III)-thiol complexes out of the cytosol. These include Mrp1 and Mrp2 in mammals that extrude As(GS)₃ into blood or bile (2), Ycf1p in yeast that extrudes As(GS)₃ into the vacuole (3), and PgpA in Leishmania that extrudes the As(III)-trypanothione complex into intracellular compartments (4). These pumps are generalized resistance pumps and are not specific for arsenite. In contrast, ArsB, the first identified member of the second family of arsenite efflux proteins, has the physiological role of conferring resistance to inorganic As(III) and Sb(III) (5, 6). The best characterized member of the ArsB family is that encoded by the arsRDABC operon of the conjugative R-factor R773 of Escherichia coli. ArsB is widespread in bacteria and archaea. It has 12 membrane-spanning segments (7), which is similar to members of the Major Facilitator Superfamily (8). It transports As(III) but has higher affinity for Sb(III). ArsB is an antiporter that catalyzes the exchange of trivalent metalloid for protons, coupling arsenite efflux to the electrochemical proton gradient (9).The third arsenic resistance transporter is Acr3, which is a member of the BART (bile/arsenite/riboflavin transporter) superfamily and includes members found in bacteria, archaea, and fungi and is more widely distributed than members of the ArsB family (10) (supplemental Fig. 1). Homologues have recently been identified in plant (Pteris vittata, NCBI accession number {"type":"entrez-protein","attrs":{"text":"ACN65413","term_id":"224814384","term_text":"ACN65413"}}ACN65413) and animal genomes (Danio rerio, NCBI accession number {"type":"entrez-protein","attrs":{"text":"XP_001921075","term_id":"189546053","term_text":"XP_001921075"}}XP_001921075). Unfortunately, the literature is confused by the fact that many members of the Acr3 family are annotated as ArsB even though they exhibit no significant sequence similarity to ArsB. The first identified member of this family is encoded by the ars operon of the skin (sigK intervening) element in the chromosome of Bacillus subtilis (11). The membrane topology of the B. subtilis Acr3 was recently investigated using translational fusions, but the results could not distinguish between 8 and 10 transmembrane-spanning segments (TMs)² (12). Fungal members of this family include the Saccharomyces cerevisiae Acr3p metalloid efflux protein (3, 13). Interestingly, yeast Acr3p appears to be selective for As(III) over Sb(III), which is surprising considering the similarity in chemical properties between the two metalloids. The properties of a more distant homologue from Shewanella oneidensis was examined recently (14). The S. oneidensis homologue confers resistance to arsenate but not arsenite. Similarly, the purified protein binds arsenate, not arsenite, indicating that this protein is not an Acr3 orthologue.Here we examined the properties of Acr3 orthologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum (supplemental Fig. 1). A. metalliredigens is a borate-tolerant Gram-positive alkaliphile and strict anaerobe that uses reduction of metals as electron acceptors (15). It is a novel metal-reducing bacterium that is distantly related to other commonly studied iron-reducing microorganisms. The genome of A. metalliredigens QYMF (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_009633","term_id":"150387853","term_text":"NC_009633"}}NC_009633) contains two novel ars operons, arsR₁B_acr3–1D₁A_1–1A_1–2 and arsR₂CB_acr3–2D₂A_2–1A_2–2. The two genes for the AmAcr3s were designated ars_acr3 because they are both in ars operons and are controlled by ArsR repressors, even though they are not homologues of ArsB. Interestingly, both ars operons have genes for ArsD and two genes corresponding to the two homologous halves of ArsA, which we designate AmArsA1 and AmArsA2. ArsD is an arsenic chaperone that transfers As(III) to ArsA (16), which then interacts with ArsB to extrude As(III) from the cells in an ATP-dependent manner (6, 17, 18). Whether or how Acr3 can replace ArsB in this process is a question of considerable interest.C. glutamicum is a Gram-positive soil bacterium that is used for commercial production of glutamate, lysine, and other amino acids, nucleotides, and vitamins and from which the genome sequence has been described (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_006958","term_id":"62388892","term_text":"NC_006958"}}NC_006958). It is highly arsenic-resistant and has three genes encoding Acr3 homologues (19). Two of the homologues are in ars operons regulated by ArsRs (arsR₁B_acr3–1C₁C_1′ and arsR₂B_acr3–2arsC₂) and a third orphan gene (arsB_acr3–3) that is not in an operon and may not be expressed to the same extent as the other two. (Again, the genes were misnamed arsB even though they encode Acr3 homologues.)The genes for AmAcr3 and CgAcr3 from the ars1 operons of the respective species were cloned and expressed in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon had been deleted (20). Both conferred resistance to arsenite but not arsenate or antimonite. Examination of the sequence of Acr3 homologues from many species indicates that there is conserved cysteine residues, Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 (supplemental Fig. 1). Those and other nonconserved cysteine residues were changed by mutagenesis, and substitution of only Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 led to loss of function, suggesting that the conserved cysteine residue participates in As(III) transport. A scanning cysteine accessibility method (SCAM) (21) was used to determine the transmembrane topology of AmAcr3. SCAM analysis is preferable to the use of gene fusions because there are minimal structural changes in the membrane protein, and the sidedness of inserted cysteines can be unambiguously determined with maleimide reagents of differing membrane permeability. A series of single cysteine mutants of AmAcr3 was constructed and the reactivity of each cysteine residue assayed. The results unambiguously demonstrate that Acr3 has 10 TMs.     

Phosphorylation of a Novel Cytoskeletal Protein (RsmP) Regulates Rod-shaped Morphology in Corynebacterium glutamicum

Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.     

Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats

Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and L?wenstein-Jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection.