Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. CT13 under abiotic stress

Polyhydroxybutyrate (PHB), a typical carbon and energy storage compound, is widely found in Bacteria and Archae domains. This polymer is produced in response to conditions of physiological stress. PHB is composed of repeating units of β-hydroxybutyrate (R-3HB). It has been previously shown that R-3HB functions as an osmolyte in extremophile strains. In this study, Pseudomonas sp. CT13, a halotolerant bacterium, and its PHB synthase-minus mutant (phaC) were used to analyze the chaperone role of R-3HB. The production of this compound was found to be essential to salt stress resistance and positively correlated with salt concentration, suggesting that PHB monomer acts as a compatible solute in Pseudomonas sp. CT13. R-3HB accumulation was also associated with the prevention of protein aggregation under combined salt and thermal stresses in Pseudomonas sp. CT13. Physiological concentrations of R-3HB efficiently reduced citrate synthase (CS) aggregation and stabilized the enzymatic activities of CS during thermal stress. Docking analysis of the CS/R-3HB interaction predicted the stability of this complex under physiological concentrations of R-3HB. Thus, in vivo, in vitro and in silico analyses suggest that R-3HB can act as a chemical chaperone.     

Differences between the Glycosylation Patterns of Haptoglobin Isolated from Skin Scales and Plasma of Psoriatic Patients

Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.     

Hyposialylation of neprilysin possibly affects its expression and enzymatic activity in hereditary inclusion-body myopathy muscle

Autosomal recessive hereditary inclusion-body myopathy (h-IBM) is caused by mutations of the UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase gene, a rate-limiting enzyme in the sialic acid metabolic pathway. Previous studies have demonstrated an abnormal sialylation of glycoproteins in h-IBM. h-IBM muscle shows the abnormal accumulation of proteins including amyloid-β (Aβ). Neprilysin (NEP), a metallopeptidase that cleaves Aβ, is characterized by the presence of several N-glycosylation sites, and changes in these sugar moieties affect its stability and enzymatic activity. In the present study, we found that NEP is hyposialylated and its expression and enzymatic activity reduced in all h-IBM muscles analyzed. In vitro , the experimental removal of sialic acid by Vibrio Cholerae neuraminidase in cultured myotubes resulted in reduced expression of NEP. This was most likely because of a post-translational modification consisting in an abnormal sialylation of the protein that leads to its reduced stability. Moreover, treatment with Vibrio Cholerae neuraminidase was associated with an increased immunoreactivity for Aβ mainly in the form of distinct cytoplasmic foci within myotubes. We hypothesize that, in h-IBM muscle, hyposialylated NEP has a role in hampering the cellular Aβ clearing system, thus contributing to its abnormal accumulation within vulnerable fibers and possibly promoting muscle degeneration.     

The ER-bound RING finger protein 5 (RNF5/RMA1) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis

Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of beta-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders.     

Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full-length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch-clamp recordings, calcium imaging and immunocytochemistry on slices from 8-12 days post-natal rats. In this preparation, BDNF (100-500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN-PAFs). Effect was blocked by trk antagonist K252a or anti-trkB antibody clone 47. A pre-synaptic mechanism was demonstrated after (i) patch-clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA-mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK(1) receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN-PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN-PAFs. This is the first demonstration that trkB receptors expressed by PN-PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.     

Identifying the minimal copper- and zinc-binding site sequence in amyloid-beta peptides

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-beta (Abeta) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Abeta peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Abeta(1-40), Abeta(1-16), Abeta(1-28), Abeta(5-23), and Abeta(17-40)). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His(6), His(13), and His(14)). In zinc-Abeta complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Abeta peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.