Tubulin polyglycylation in Platyhelminthes: diversity among stable microtubule networks and very late occurrence during spermiogenesis

The distribution of glycylated tubulin has been analyzed in different populations of stable microtubules in a digenean flatworm, Echinostoma caproni (Platyhelminthes). Two cellular types, spermatozoa and ciliated excretory cells, have been analyzed by means of immunofluorescence, immunogold, and immunoblotting techniques using two monoclonal antibodies (mAbs), AXO 49, and TAP 952, specifically directed against differently glycylated isoforms of tubulin. The presence of glycylated tubulin in the two cell types was shown. However, the differential reactivities of TAP 952 and AXO 49 mAbs with the two axoneme types suggest a difference in their glycylation level. In addition, within a single cell, the spermatozoon, cortical microtubules underlying the flagellar membrane, and axonemal microtubules were shown to comprise different tubulin isoforms, the latter ones only being labelled with one of the antiglycylated tubulin mAbs, TAP 952. Similarly, the antiacetylated (6-11B-1) and polyglutamylated (GT335) tubulin mAbs decorated the two types of axonemal microtubules, but not the cortical ones. From these data, a subcellular sorting of posttranslationally modified tubulin isoforms within spermatozoa, on the one hand, and a cellular sorting of glycylated isoforms inside the whole organism, on the other hand, is demonstrated in the flatworm E. caproni. Last, a sequential occurrence of tubulin posttranslational modifications was observed in the course of spermiogenesis. Acetylation appears first, followed shortly by glutamylation; glycylation takes place at the extreme end of spermiogenesis and, specifically, in a proximo-distal process. Thus in agreement with, and extending other studies [Bré et al., 1996], glycylation appears to close the sequence of posttranslational events occurring in axonemal microtubules during spermiogenesis.     

The CATH Database provides insights into protein structure/function relationships

We report the latest release (version 1.4) of the CATH protein domains database (http://www.biochem.ucl.ac.uk/bsm/cath). This is a hierarchical classification of 13 359 protein domain structures into evolutionary families and structural groupings. We currently identify 827 homologous families in which the proteins have both structual similarity and sequence and/or functional similarity. These can be further clustered into 593 fold groups and 32 distinct architectures. Using our structural classification and associated data on protein functions, stored in the database (EC identifiers, SWISS-PROT keywords and information from the Enzyme database and literature) we have been able to analyse the correlation between the 3D structure and function. More than 96% of folds in the PDB are associated with a single homologous family. However, within the superfolds, three or more different functions are observed. Considering enzyme functions, more than 95% of clearly homologous families exhibit either single or closely related functions, as demonstrated by the EC identifiers of their relatives. Our analysis supports the view that determining structures, for example as part of a 'structural genomics' initiative, will make a major contribution to interpreting genome data.     

NMR structure of the (52-96) C-terminal domain of the HIV-1 regulatory protein Vpr: molecular insights into its biological functions

The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.     

Bacterial aminopeptidases: properties and functions

Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasic activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains. At present, about one hundred bacterial aminopeptidases have been purified and biochemically studied. About forty genes encoding aminopeptidases have also been cloned and characterised. Recently, the three-dimensional structure of two aminopeptidases, the methionine aminopeptidase from Escherichia coli and the leucine aminopeptidase from Aeromonas proteolytica, have been elucidated by crystallographic studies. Most of the quoted studies demonstrate that bacterial aminopeptidases generally show Michaelis-Menten kinetics and can be placed into either of two categories based on their substrate specificity: broad or narrow. These enzymes can also be classified by another criterium based on their catalytic mechanism: metallo-, cysteine- and serine-aminopeptidases, the former type being predominant in bacteria. Aminopeptidases play a role in several important physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied peptides and are necessary for the final steps of protein turnover. In addition, they are involved in some specific functions, such as the cleavage of N-terminal methionine from newly synthesised peptide chains (methionine aminopeptidases), the stabilisation of multicopy ColE1 based plasmids (aminopeptidase A) and the pyroglutamyl aminopeptidase (Pcp) present in many bacteria and responsible for the cleavage of the N-terminal pyroglutamate.     

New insights from spectroscopy into the structure/function relationships of lipoxygenases

Spectroscopic properties of the redox-active iron in the active site of plant and mammalian lipoxygenases can now be combined with recent crystal structure determinations to obtain new insights into lipoxygenase reaction mechanisms.     

Childhood mania: insights into diagnostic and treatment issues

Our study's objective was to clarify the nosologic status of children who satisfy diagnostic criteria for bipolar disorder. Using blind raters and structured psychiatric interviews as well as data from other instruments, we undertook studies from various vantage points, examining children with bipolar disorder to confirm the existence of the diagnosis in children and to clarify its clinical course and characteristics. We found that (1) bipolar disorder in children referred to our clinical center is not as rare as previously thought; (2) bipolar disorder in children commonly presents with attention-deficit hyperactivity disorder, which makes diagnosis difficult; and (3) bipolar disorder in children presents with a clinical picture considered atypical by adult standards, with irritability, chronicity, and symptoms of mania mixed with those of depression. Our data suggest that childhood bipolar disorder is more common than previously thought, but it may be difficult to diagnose because of comorbidity with attention-deficit hyperactivity disorder and a developmentally different presentation from adults.     

Modeling elastic properties of microtubule tips and walls

In human tissues different populations of dendritic cells (DC) emerge from hematopoietic progenitor cells (HPC) in the bone marrow, with the intermediate steps of differentiation not being completely understood. In vitro, DC can be directly obtained from HPC or from blood monocytes (MO) cultured in the presence of GM-CSF and additional cytokines. We compared the antigenic profile of DC derived from either MO or HPC and studied their capacity to stimulate naive lymphocytes (LY) in the allogeneic mixed lymphocyte reaction. Both types of DC expressed high levels of CD1a, MHC class II, CD80, CD86 and CD40 and were potent stimulators of LY proliferation. DC of HPC origin, though, induced a stronger mixed lymphocyte reaction than MO-derived DC and showed a slightly higher average expression of costimulatory antigens. Low-level expression of CD14 did not negatively correlate with DC function on DC stimulated with lipopolysaccharide and was even slightly higher expressed on DC differentiating from HPC than on DC from CD14+ MO.     

A cohesin domain from Clostridium thermocellum: the crystal structure provides new insights into cellulosome assembly

The most common major comorbid disorders associated with eating disorders include substance use disorders, personality disorders, mood disorders, anxiety disorders, and obesity. To test conceptual models of the relationship between the eating disorders and these comorbid disorders, complex research paradigms are needed, including epidemiological studies, behavior-genetic studies, and longitudinal research designs. Comorbidity may be a significant factor to consider as approaches to the treatment of eating disorders continue to evolve.     

Molecular insights into cancer invasion: strategies for prevention and intervention

The diagnosis and treatment of solid tumors usually begins at a late stage when most patients already have occult or overt metastasis. Many years of cancer progression precede diagnosis of most solid tumors. Novel noncytotoxic therapeutics may be specially suited for administration during this interval. An important window of intervention can be defined as the period during which transition from a hyperproliferative state to acquisition of the capacity for invasion and metastasis occurs. Investigation of the molecular basis of invasion is uncovering strategies for delaying progression of preinvasive carcinoma and treatment of primary tumors and established metastasis. Although tumor cell invasion might not be rate limiting for the growth of metastasis, anti-invasive agents can block tumor angiogenesis and thereby indirectly block metastasis growth. Two classes of molecular anti-invasion targets exist: (a) cell surface and extracellular proteins, which mediate sensing, adhesion, and proteolysis; and (b) signal transduction pathways, which regulate invasion, angiogenesis, and proliferation. Both categories of targets yield treatment approaches that are now being tested in the clinic. Metalloproteinase inhibitors, such as BB94, are based on the recognition that metalloproteinases play a necessary role in invasion and angiogenesis. The orally active signal transduction inhibitor carboxyamidotriazole modulates non-voltage-gated calcium influx-regulated signal pathways and reversibly inhibits tumor invasion, growth, and angiogenesis. Blockade of invasion, angiogenesis, or cellular signal pathways is likely to generate a cytostatic, rather than a cytotoxic effect. Cytostatic therapy constitutes an alternative paradigm for clinical translation that may complement conventional cytotoxic therapy. For patients with newly diagnosed solid tumors, long-term cytostatic therapy could potentially create a state of metastasis dormancy or delay the time to overt relapse following cytotoxic agent-induced remission. Clinical toxicity and pharmacology using oral cytostatic agents in phase I trials and in adjuvant settings will provide an important foundation for the translation of this approach to the preinvasive carcinoma period.     

Clinical insights into pragmatic theory: frontal lobe deficits and sarcasm

The validity of psycholinguistic theories of sarcasm was explored by examining subjects with mainly frontal lobe (FL) damage and concomitant concreteness of thought. The majority of FL subjects could interpret consistent verbal exchanges but not literally contradictory (sarcastic) verbal exchanges which implied that the literal meaning of a sarcastic comment needs to be rejected in order for the inference to be detected. Subsidiary analyses confirmed that failure on sarcasm tasks was associated with poor conceptual skills. Ability to process attitude was not associated with success at recognizing sarcasm. This suggested that attitude is not pivotal to the detection of sarcastic inference.     

The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance

BACKGROUND:. The ribosomal protein L22 is one of five proteins necessary for the formation of an early folding intermediate of the 23S rRNA. L22 has been found on the cytoplasmic side of the 50S ribosomal subunit. It can also be labeled by an erythromycin derivative bound close to the peptidyl-transfer center at the interface side of the 50S subunit, and the amino acid sequence of an erythromycin-resistant mutant is known. Knowing the structure of the protein may resolve this apparent conflict regarding the location of L22 on the ribosome. RESULTS:. The structure of Thermus thermophilus L22 was solved using X-ray crystallography. L22 consists of a small alpha+beta domain and a protruding beta hairpin that is 30 A long. A large part of the surface area of the protein has the potential to be involved in interactions with rRNA. A structural similarity to other RNA-binding proteins is found, possibly indicating a common evolutionary origin. CONCLUSIONS:. The extensive surface area of L22 has the characteristics of an RNA-binding protein, consistent with its role in the folding of the 23S rRNA. The erythromycin-resistance conferring mutation is located in the protruding beta hairpin that is postulated to be important in L22-rRNA interactions. This region of the protein might be at the erythromycin-binding site close to the peptidyl transferase center, whereas the opposite end may be exposed to the cytoplasm.     

Subunit epsilon of the Escherichia coli ATP synthase: novel insights into structure and function by analysis of thirteen mutant forms

Structural models of subunit epsilon of the ATP synthase from Escherichia coli have been determined recently by NMR [Wilkens et al. (1995) Nat. Struct. Biol. 2, 961-967] and by X-ray crystallography [Uhlin et al. (1997) Structure 5, 1219-1230], revealing a two-domain protein. In this study, six new epsilon mutants were constructed and analyzed: Y63A, D81A, T82A, and three truncated mutants, tr80(S), tr94(LAS), and tr117(AS). Seven mutants constructed previously were also analyzed: E31A, E59A, S65A, E70A, T77A, R58A, and D81A/R85A. Subunits were purified by isoelectric focusing from extracts of cells that overproduced these 13 mutants. F1 was prepared lacking subunit epsilon by immobilized-Ni affinity chromatography. Three mutants, E70A, S65A, and E31A, showed somewhat higher affinities and extents of inhibition than the wild type. Three mutants, T82A, R85A, and tr94(LAS), showed both lower affinities and extents of inhibition, over the concentration range tested. Two showed no inhibition, D81A and tr80(S). The others, T77A, Y63A, E59A, and tr117(AS), showed lower affinities than wild type, but the extents of inhibition were nearly normal. Results indicate that the C-terminal domain of subunit epsilon contributes to inhibition of ATP hydrolysis, but it is not necessary for ATP-driven proton translocation. Interactions with subunit gamma are likely to involve a surface containing residues S65, E70, T77, D81, and T82, while residues R85 and Y63 are likely to be important in the conformation of subunit epsilon.     

Molecular insights into renal interstitial fibrosis

Progressive interstitial fibrosis accompanied by loss of renal tubules and interstitial capillaries typifies all progressive renal diseases. Dynamic and complex, the process evidently overlaps with matrix remodeling; it may even be reversible. The interstitial fibrous tissue comprises several normal and novel matrix proteins, proteoglycans, and glycoproteins. Interstitial myofibroblasts are a major site of matrix protein overproduction, although resident fibroblasts, tubular cells, and inflammatory cells may contribute. Inadequate matrix degradation also appears to contribute to the fibrogenic process. Two protease cascades, the metalloproteinases and the plasminogen activator/ plasmin family of serine proteases, are implicated in the turnover of interstitial matrix proteins; upregulated expression of protease inhibitors has been observed in each. Increased tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 levels suggest that the intrinsic renal activity of the metalloproteinases and serine proteases are inhibited while matrix proteins accumulate in the interstitium. Several signals that may direct the interstitial fibrogenic process have been identified, but not yet proved to cause it. Upregulated expression of transforming growth factor beta-1, the proteotypic fibrogenic cytokine, has been observed in experimental and human models; it probably does not act alone. There may be supportive roles for platelet-derived growth factor, interleukin-1, basic fibroblast growth factor, angiotensin II, and endothelin-1. Although it is not known why interstitial fibrosis compromises renal function, atrophy of renal tubules may be pivotal. Ischemic necrosis and/or apoptosis may generate nonfunctioning atubular and sclerotic glomeruli. Future studies must delineate the molecular basis of the differences between renal repair and renal destruction by fibrosis, two processes that share many common features.     

Molecular genetic insights into cardiovascular disease

The recent application of molecular genetic tools to inherited forms of cardiovascular disease has provided important insight into the molecular mechanisms underlying cardiac arrhythmias, cardiomyopathies, and vascular diseases. These studies point to defects in ion channels, contractile proteins, structural proteins, and signaling molecules as key players in disease pathogenesis. Genetic testing is now available for a subset of inherited cardiovascular diseases, and new mechanism-based therapies may be available in the near future. This remarkable progress and the implications it may have for more common forms of cardiovascular disease are reviewed here.