Protease-catalysed protein splicing: a new post-translational modification?

In all organisms, proteases catalyse peptide-bond hydrolysis and mediate protein function for a multitude of cellular processes. Mechanistically, nothing prevents proteases from also catalysing peptide-bond ligation; however, this 'reverse' reaction rarely is observed. In eukaryotes its presence has been viewed as an anomaly. Recent studies from plants and animals now challenge this assumption, indicating that protease-catalysed protein splicing is a bona fide post-translational modification. Increasing evidence indicates that the proximity of protein substrates, imposed either by their structure or by the physical constraints of the local environment, dictates when the splicing reaction will occur. This previously under-recognized splicing mechanism could increase intracellular protein diversity, thereby expanding the size of the proteome and sequence diversity beyond the predictions from genomic studies.     

Tyrosine sulfation: a post-translational modification of proteins destined for secretion?

Protein sulfation was studied in germ-free rats by prolonged in vivo labeling with [35S]sulfate. Specific sets of sulfated proteins were observed in all tissues examined, in leucocytes, and in blood plasma. No protein sulfation was detected in erythrocytes. Analysis of the type of sulfate linkage showed that sulfated proteins secreted into the plasma contained predominantly tyrosine sulfate, whereas sulfated proteins found in tissues contained largely carbohydrate sulfate. This implies some kind of selection concerning the intracellular processing, secretion, turnover or re-uptake of sulfated proteins which is responsible for the enrichment of tyrosine-sulfated proteins in the plasma.     

Hsp90 regulates O-linked β-N-acetylglucosamine transferase: a novel mechanism of modulation of protein O-linked β-N-acetylglucosamine modification in endothelial cells

O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is involved in many important cellular processes. Increased O-GlcNAc has been implicated in major diseases, such as diabetes and its complications and cardiovascular and neurodegenerative diseases. Recently, we reported that O-GlcNAc modification occurs in the proteasome and serves to inhibit proteasome function by blocking the ATPase activity in the 19S regulatory cap, explaining, at least in part, the adverse effects of O-GlcNAc modification and suggesting that downregulating O-GlcNAc might be important in the treatment of human diseases. In this study, we report on a novel mechanism to modulate cellular O-GlcNAc modification, namely through heat shock protein 90 (Hsp90) inhibition. We observed that O-linked β-N-acetylglucosamine transferase (OGT) interacts with the tetratricopeptide repeat binding site of Hsp90. Inhibition of Hsp90 by its specific inhibitors, radicicol or 17-N-allylamino-17-demethoxygeldanamycin, destabilized OGT in primary endothelial cell cultures and enhanced its degradation by the proteasome. Furthermore, Hsp90 inhibition downregulated O-GlcNAc protein modifications and attenuated the high glucose-induced increase in O-GlcNAc protein modification, including high glucose-induced increase in endothelial or type 3 isoform of nitric oxide synthase (eNOS) O-GlcNAcylation. These results suggest that Hsp90 is involved in the regulation of OGT and O-GlcNAc modification and that Hsp90 inhibitors might be used to modulate O-GlcNAc modification and reverse its adverse effects in human diseases.     

The ins and outs of tubulin acetylation: more than just a post-translational modification?

Microtubules are highly dynamic polymers of α/β tubulin heterodimers that play key roles in cell division and in organizing cell cytoplasm. Although they have been discovered more than two decades ago, tubulin post-translational modifications recently gained a new interest as their role was increasingly highlighted in neuron differentiation and neurodegenerative disorders. Here, we specifically focus on tubulin acetylation from its discovery to recent studies that provide new insights into how it is regulated in health and disease and how it impacts microtubule functions. Even though new mechanisms involving tubulin acetylation are regularly being uncovered, the molecular links between its location inside the microtubule lumen and its regulators and effectors is still poorly understood. This review highlights the emerging roles of tubulin acetylation in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signalling. It also points out that tubulin acetylation should no longer be seen as a passive marker of microtubule stability, but as a broad regulator of microtubule functions.     

A novel type of energetics in a marine alkali-tolerant bacterium: Δμ-Na-driven motility and sodium cycle

Motility of a marine alkali-tolerant bacterium, Vibrio alginolyticus, can be observed in the presence of high concentrations of a protonophorous uncoupler, CCCP. Motility in the CCCP-containing media is completely inhibited by decrease in extracellular [Na⁺] or by monensin-induced increase in intracellular [Na⁺]. A mutant has been selected that grows only in media supplemented with a substrate such as acetate requiring no Δ$\text{μ}\text{-}$_Na to be transported into the cell. Motility of the mutant was found to be completely inhibited by CCCP. Cyanide, CCCP and vanadate added separately or in twos inhibit motility only partially. The three poisons added together completely paralyse the cells. In this inhibitor cocktail, arsenate can substitute for CCCP + vanadate; cyanide can be replaced by anaerobiosis. It is concluded that (i) Δ$\text{μ}\text{-}$_Na rather than Δ$\text{μ}\text{-}$_NH powers the flagellar motor of V. alginolyticus in the presence of CCCP, and (ii) in addition to the Na⁺-motive respiratory chain [Tokuda, H. and Unemoto, T. (1982) J. Biol. Chem. 257, 10007–10014] there is a vanadate and arsenate-sensitive oxygen-independent mechanism of Δ$\text{μ}$_Na generation, presumably an ion-motive ATPase. A suggestion is put forward that circulation of Na⁺ can replace that of H⁺ in V. alginolyticus, Δ$\text{μ}\text{-}$_Na being formed by the Na⁺-motive respiratory chain and utilized by Na⁺-solute symporters, the Na⁺-driven flagellar motor and maybe by a reverse ion-motive ATPase.     

Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.     

Structures of the tumor necrosis factor α inducing protein Tipα: A novel virulence factor from Helicobacter pylori

Helicobacter pylori secretes a unique virulence factor, Tipα, that enters gastric cells and both stimulates the production of the TNF-α and activates the NF-κB pathway. The structures of a truncated version of Tipα (TipαN34) in two crystal forms are presented here. Tipα adopts a novel β₁α₁α₂β₂β₃α₃α₄ topology that can be described as a combination of three domains. A first region consists in a short flexible extension, a second displays a dodecin-like fold and a third is a helical bundle domain similar to the sterile alpha motif (SAM). Analysis of the oligomerisation states of TipαN34 in the crystals and in solution suggests that the disulfide bridges could hold together Tipα monomers during their secretion in the gastric environment.

Structured summary

MINT-7033680:TIP alpha (uniprotkb:B2UTN0) and TIP alpha (uniprotkb:B2UTN0) bind (MI:0407) by cosedimentation (MI:0027)     

A novel RAB7 mutation in a Chinese family with Charcot–Marie–Tooth type 2B disease

Charcot–Marie–Tooth type 2B (CMT2B) disease is a hereditary motor and sensory neuropathy subtype characterized by prominent loss of sensation, distal muscle weakness and wasting skin ulcers. Recurrent ulcers often require amputation of lower limbs. To date, only four mutations of the RAB7 gene, which encodes the small GTPase, have been associated with CMT2B. A Chinese family with CMT2B was identified. Direct DNA sequencing performed on the affected individuals in this family revealed a novel mutation (p.Asn161Ile) in RAB7. The mutation is located in a potential mutational hotspot region, implicating the importance of this region for RAB7 protein. This is the first report of RAB7 mutation in Asian population.     

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA_A receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA_A receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA_A receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA_A receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA_A receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA_A receptor subtypes and might contribute to anchoring and/or confining GABA_A receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.     

A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4–1.2 pH units, likely due to repositioning of the cofactor and modification of the pK_a of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3–3, 26–58 and 124–568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The K_m^l-Cth of E333 substitution variants is increased ~ 17-fold, while K_m^l-OAS is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (k_cat/K_m^l-OSHS = 7 ± 2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (k_cat/K_m^l-Cth = 2100 ± 100) and 260-fold higher than that of l-Hcys (k_cat/K_m^l-Hcys = 0.027 ± 0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.     

Chemical modification of Interleukin-1β: Biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein

We have modified recombinant human Interleukin-1 using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide atpH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI=6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys₈₈ and Lys₉₂). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu₁₁₁ and Lys₁₃₈ were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1 and may be important for the binding of IL-1 to its receptor.     

Silibinin: a novel inhibitor of Aβ aggregation

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β peptide (Aβ) into extracellular fibrillar deposits known as amyloid plaque. Inhibition of Aβ aggregation is therefore viewed as a potential method to halt or slow the progression of AD. It is reported that silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), attenuates cognitive deficits induced by Aβ25-35 peptide and methamphetamine. However, it remains unclear whether silibinin interacts with Aβ peptide directly and decreases Aβ peptide-induced neurotoxicity. In the present study, we identified, through employing a ThT assay and electron microscopic imaging that silibinin also appears to act as a novel inhibitor of Aβ aggregation and this effect showed dose-dependency. We also show that silibinin prevented SH-SY5Y cells from injuries caused by Aβ(1-42)-induced oxidative stress by decreasing H(2)O(2) production in Aβ(1-42)-stressed neurons. Taken together, these results indicate that silibinin may be a novel therapeutic agent for the treatment of AD.     

Identification of a novel ZNF469 mutation in a large family with Ehlers–Danlos phenotype

Brittle cornea syndrome (BCS) is a genetically heterogeneous disorder characterized by extreme corneal fragility and thinning, which may lead to spontaneous or trauma-induced corneal rupture. BCS-1 and BCS-2 are caused by recessive mutations in ZNF469 and PRDM5, respectively. Both genes play a role in the regulatory pathway of corneal development and maintenance. We report a consanguineous family with five patients affected with the cardinal ocular features of BCS and significant musculoskeletal findings primarily in the form of joint hypermobility and severe kyphoscoliosis. The patients had thin velvety skin, hallux valgus, variable sensorineural hearing loss and arachnodactyly. Interestingly, one of the patients additionally had phenylketonuria and showed a milder ophthalmological and musculoskeletal phenotype than his affected siblings. The urinary pyridinoline and deoxypyridinoline concentrations and their ratios were mildly elevated indicating increased bone-collagen turnover. A novel homozygous 14 bp duplication in exon 2 of ZNF469 (c.8817_8830dup) was uncovered by direct sequencing. This family highlights the phenotypic overlap between BCS and Ehlers–Danlos syndrome.     

Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis

Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent bone-resorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.