Composition and organization of tubulin isoforms reveals a variety of axonemal models

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.     

5-methylthioribose 1-phosphate: a product of partially purified, rat liver 5'-methylthioadenosine phosphorylase activity.

5'-Methylthioadenosine phosphorylase from rat liver has been purified 112-fold. A molecular weight of 90 000 for the enzyme was estimated from gel filtration on Sephadex G-150. The Km for 5'-methylthioadenosine was 4.7 . 10(-7) M, while the Km for phosphate was 2 . 10(-4) M. The products of the reaction were isolated and identified as adenine and 5-methylthioribose 1-phosphate. In addition to 5'-methylthioadenosine the nucleoside analogues 5'-ethylthioadenosine and 5'-n-propylthioadenosine also served as substrates for the enzyme. The 7-deaza analogue 5'-methylthiotubercidin was found to be an inhibitor of the reaction, but was inactive as a substrate.     

Hydroxysteroid dehydrogenase transformations of 5β-scymnol and identification of oxoscymnol transformation products by liquid chromatography-tandem mass spectroscopy

A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS(2)) detection was developed to identify for the first time the oxidation products of 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H](+) at m/z 469 Da, and higher mass adduct ions attributed to [M+NH(4)](+) (m/z 486), [M+H+CH(3)OH](+) (m/z 501) and [M+H+CH(3)COOH](+) (m/z 530). (24R)-(+)-5β-Cholestan-3-one-7α,12α,24,26,27-pentol (3-oxoscymnol, m/z 467 Da, relative retention time (RRT)=0.89) was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. [S](0.5) for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5β-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m/z 467 Da, RRT=0.79) and (24R)-(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m/z 467 Da, RRT=0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol>12-oxoscymnol>3-oxoscymnol>scymnol (in order from most polar to least polar). Confirmation that 5β-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography-mass spectrometry (LC-MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.     

Lysosomal peptidases and glycosidases in rheumatoid arthritis

Lysosomal serine and cysteine proteases are reported to play a role in collagen degradation. In this study, the activities of the lysosomal cysteine proteases cathepsin B and H, dipeptidyl peptidase I, and the serine protease tripeptidyl peptidase I and dipeptidyl peptidase II, all ascribed a role in collagen digestion, were compared with those of the aspartate protease cathepsin D, and lysosomal glycosidases in leukocytes from rheumatoid arthritis patients at different stages of the disease. In all patients the activities of cysteine protease cathepsin B, dipeptidyl peptidase I, aspartate protease cathepsin D, and two glycosidases were elevated, but the activities of the serine proteases tripeptidyl peptidase I, dipeptidyl peptidase II, and the cysteine protease cathepsin H was unchanged. The magnitude of the increased activity was correlated with the duration of the disease. Patients with long-standing RA (10 years or more) had higher cysteine protease activity in their leukocytes than did those with disease of shorter duration. This tendency suggests that elevated lysosomal cysteine protease activities, together with aspartate protease cathepsin D and lysosomal glycosidases (but not serine proteases), are associated with progression of rheumatoid arthritis.     

Where and when is microtubule diversity generated inParamecium? Immunological properties of microtubular networks in the interphase and dividing cells

Summary Ciliates are highly differentiated cells which display extensive deployment of microtubular systems. Because genetic diversity of tubulin is extremely reduced in these cells, microtubule diversity is mostly generated at the post-translational level either through direct modification of tubulin or through the binding of associated proteins to microtubules. We have undertaken a systematic exploration of microtubule diversity in ciliates by way of production of monoclonal antibodies. Previously we reported the biochemical characterization of these antibodies. In addition to antibodies directed against primary sequences, we obtained antibodies directed against post-translational modifications. In this paper, we report a detailed analysis of the distribution of the various epitopes on the microtubular networks ofParamecium, both in interphase cells and during division morphogenesis. Each of these antibodies decorates a subset of microtubules. Acetylation, recognized by antibodies TEU 318 and TEU 348, is detected on stable microtubules early after microtubule assembly. Epitopes recognized by two other antibodies (TAP 952 and AXO 58) are found on a subset of stable microtubules; in addition, the TAP 952 antibody is also found on labile microtubules; both epitopes are detected as soon as microtubule assembly occurs. In contrast, the epitope of the antibody, AXO 49, is associated with only a restricted subset of stable microtubules in the interphase cell, and is detected a lag-time after microtubule assembly during division morphogenesis. These data show that microtubule diversity is generated through a time-dependent sequence and according to a definite spatial pattern.     

sHSP in the eye lens: crystallin mutations, cataract and proteostasis

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin "holdase" chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the "α-crystallin domain" of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens β- and γ-crystallins are considered in the context of their possible dependence on the "holdase" chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.     

Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.     

Reactions of some cyclopentenones with selected cysteine derivatives and biological activities of the product thioethers

The conjugate addition reaction between glutathione, N-Boc-cysteine methyl ester, N-acetyl cysteine methyl ester and N-acetyl cysteine and several substituted cyclopentenones is described. The reversibility of this process was demonstrated by thio-adduct metathesis on treatment of the adduct with a different cysteinyl derivative. The levels at which these compounds inhibit the function of nuclear factor kappa B (NF-kappaB) and potentiate heat shock factor (HSF) are reported and the possible relevance of these studies concerning the antiviral and anti-inflammatory activities of the cyclopentenone prostanoids is discussed.