Spastin regulates VAMP7-containing vesicles trafficking in cortical neurons.

Alteration of axonal transport has emerged as a common precipitating factor in several neurodegenerative disorders including Human Spastic Paraplegia (HSP). Mutations of the SPAST (SPG4) gene coding for the spastin protein account for 40% of all autosomal dominant uncomplicated HSP. By cleaving microtubules, spastin regulates several cellular processes depending on microtubule dynamics including intracellular membrane trafficking. Axonal transport is fundamental for the viability of motor neurons which often have very long axons and thus require efficient communication between the cell body and its periphery. Here we found that the anterograde velocity of VAMP7 vesicles, but not that of VAMP2, two vesicular-SNARE proteins implicated in neuronal development, is enhanced in SPG4-KO neurons. We showed that this effect is associated with a slight increase of the level of acetylated tubulin in SPG4-KO neurons and correlates with an enhanced activity of kinesin-1 motors. Interestingly, we demonstrated that an artificial increase of acetylated tubulin by drugs reproduces the effect of Spastin KO on VAMP7 axonal dynamics but also increased its retrograde velocity. Finally, we investigated the effect of microtubule targeting agents which rescue axonal swellings, on VAMP7 and microtubule dynamics. Our results suggest that microtubule stabilizing agents, such as taxol, may prevent the morphological defects observed in SPG4-KO neurons not simply by restoring the altered anterograde transport to basal levels but rather by increasing the retrograde velocity of axonal cargoes.

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

TRAM domain proteins present in Archaea and Bacteria have a ß-barrel shape with anti-parallel ß-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

Tertiary structure of plant RuBisCO: domains and their contacts.

The three-dimensional structure of ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO), has been determined at 2.6 A resolution. This enzyme initiates photosynthesis by combining carbon dioxide with ribulose bisphosphate to form two molecules of 3-phosphoglycerate. In plants, RuBisCO is built from eight large (L) and eight small (S) polypeptide chains, or subunits. Both S chains and the NH2-terminal domain (N) of L are antiparallel beta, "open-face-sandwich" domains with four-stranded beta sheets and flanking alpha helices. The main domain (B) of L is an alpha/beta barrel containing most of the catalytic residues. The active site is in a pocket at the opening of the barrel that is partly covered by the N domain of a neighboring L chain. The domain contacts of the molecule and its conserved residues are discussed in terms of this structure.

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.

Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii.

Cold environments dominate the Earth's biosphere and the resident microorganisms play critical roles in fulfilling global biogeochemical cycles. However, only few studies have examined the molecular basis of thermosensing; an ability that microorganisms must possess in order to respond to environmental temperature and regulate cellular processes. Two component regulatory systems have been inferred to function in thermal regulation of gene expression, but biochemical studies assessing these systems in Bacteria are rare, and none have been performed in Archaea or psychrophiles. Here we examined the LtrK/LtrR two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii, assessing kinase and phosphatase activities of wild-type and mutant proteins. LtrK was thermally unstable and had optimal phosphorylation activity at 10 °C (the lowest optimum activity for any psychrophilic enzyme), high activity at 0 °C and was rapidly thermally inactivated at 30 °C. These biochemical properties match well with normal environmental temperatures of M. burtonii (0-4 °C) and the temperature this psychrophile is capable of growing at in the laboratory (-2 to 28 °C). Our findings are consistent with a role for LtrK in performing phosphotransfer reactions with LtrR that could lead to temperature-dependent gene regulation.

Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.

No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.

Low frequency of TSG101/CC2 gene alterations in invasive human breast cancers.

Large intragenic deletions of the TSG101/CC2 gene were recently reported in seven of 15 primary metastatic breast cancers. Although the number of samples was small, this observation suggested that TSG101/CC2 alterations were a major event in breast carcinogenesis. To study the frequency of these deletions in invasive breast cancers we analysed 189 primary invasive breast tumours and 59 breast cancer metastases. We detected intragenic rearrangements in only three samples (two primary tumours and one metastasis). Northern blot analysis of 43 tumours without rearrangements failed to detect any abnormalities. Furthermore, we studied TSG101/CC2 in 11 human breast adenocarcinoma cell lines by Southern blot, RT-PCR and sequencing of the entire coding region of the gene, and detected no abnormalities. These results show that genetic alteration of TSG101/CC2 is a rare event in breast cancer.

Crystalline actin tubes. III. The interaction of scandium and yttrium with skeletal muscle actin.

The effect of the trivalent cations scandium (Sc3+) and yttrium (Y3+) on the conformation of G-actin was examined using ultraviolet difference and high resolution 1H-NMR spectroscopy. A comparison was made with data obtained previously with the trivalent lanthanide cations (Ln3+). These results indicate that the first and subsequent Y ions (ionic radius 101.9 pm) behave exactly like Ln3+. Sc3+ is a smaller ion (87 pm) than any of the Ln3+. The first Sc3+ binds to a site on actin that is inaccessible to Mg2+, Y3+ and Ln3+. However, the second Sc3+ to bind behaves like an Ln3+. On replacing the native divalent cation (Mg2+), both Y3+ and Sc3+ mobilize the adenine ring of ATP bound to actin, thus exposing underlying residues to the solvent. When Y3+ and Sc3+ saturate their binding sites on actin, and when the ionic strength is raised to 0.1 M with KCl at pH 6.9, the actin aggregates. Y3+ binds to actin with a ratio of 6 : 1 and induces the aggregation of actin into crystalline actin tubes, whilst Sc3+ binds with a ratio of 8 : 1 and induces amorphous actin aggregates. These results are consistent with the suggestion that actin tubes are induced by trivalent cations, principally on the basis of their binding stoichiometry, which is determined by ionic radius.

A re-investigation of actin monomer conformation under polymerizing conditions based on rates of enzymatic digestion and ultraviolet difference spectroscopy.

There have been several reports which describe a conformational change of G-actin monomer in the presence of 0.1 M KCl. This altered monomer has been variously named, depending on whether the authors believed that it resembles G-actin, F-actin or has a conformation of its own. In this report we re-examine the experimental evidence for these proposals. The techniques include measurements of the rates of proteolytic digestion as well as near and far ultraviolet difference spectroscopy of actin in the presence and absence of KCl. We conclude that there is no compelling evidence for proposing a novel form of actin monomer.

The transition between the open and closed states of rubisco is triggered by the inter-phosphate distance of the bound bisphosphate.

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyses the central CO(2)-fixing reaction of photosynthesis in a complex, multiple-step process. Several structures of rubisco complexed with substrate analogues, inhibitors and products have been determined by X-ray crystallography. The structures fall into two well-defined and distinct states. The active site is either "open" or "closed". The timing and mechanism of the transition between these two states have been uncertain. We solved the crystal structure of unactivated (metal-free) rubisco from tobacco with only inorganic phosphate bound and conclude that phosphate binding per se does not trigger closure, as it does in the similarly structured enzyme, triosephosphate isomerase. Comparison of all available rubisco structures suggests that, instead, the distance between the terminal phosphates (P1 and P2) of the bisphosphate ligand is the trigger: if that distance is less than 9.1 A, then the active site closes; if it is greater than 9.4 A then the enzyme remains open. Shortening of the inter-phosphate distance results from the ligand binding in a more curved conformation when O atoms of the ligand's sugar backbone interact either with the metal, if it is present, or with charged groups in the metal-binding site, if the metal is absent. This shortening brings the P1 phosphate into hydrogen bonding contact with Thr65. Thr65 exists in two discrete states related by a rotation of the backbone psi torsion angle. This rotation is coupled to domain rotation and hence to active site closure. Rotation of the side-chain of Thr65 also affects the C-terminal strand of large subunit which packs against Loop 6 after closure. The position of the C-terminal strand in the closed state is stabilised by multiple polar interactions with a distinctive highly-charged latch site involving the side-chain of Asp473. In the open state, this latch site may be occupied instead by phosphorylated anions.

Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration.

The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.

Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs.

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.

Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability.

Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild-type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure.

Crystal structure of activated tobacco rubisco complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate.

The crystal structure of activated tobacco rubisco, complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate (CABP) has been determined by molecular replacement, using the structure of activated spinach rubisco (Knight, S., Andersson, I., & Brändén, C.-I., 1990, J. Mol. Biol. 215, 113-160) as a model. The R-factor after refinement is 21.0% for 57,855 reflections between 9.0 and 2.7 A resolution. The local fourfold axis of the rubisco hexadecamer coincides with a crystallographic twofold axis. The result is that the asymmetric unit of the crystals contains half of the L8S8 complex (molecular mass 280 kDa in the asymmetric unit). The activated form of tobacco rubisco is very similar to the activated form of spinach rubisco. The root mean square difference is 0.4 A for 587 equivalent C alpha atoms. Analysis of mutations between tobacco and spinach rubisco revealed that the vast majority of mutations concerned exposed residues. Only 7 buried residues were found to be mutated versus 54 residues at or near the surface of the protein. The crystal structure suggests that the Cys 247-Cys 247 and Cys 449-Cys 459 pairs are linked via disulfide bridges. This pattern of disulfide links differ from the pattern of disulfide links observed in crystals of unactivated tobacco rubisco (Curmi, P.M.G., et al., 1992, J. Biol. Chem. 267, 16980-16989) and is similar to the pattern observed for activated spinach tobacco.

Automated protein design and sequence optimisation: scoring functions and the search problem.

Advances in molecular biology may mean that almost any protein sequence can be synthesised, but perhaps this has served to highlight the inadequacy of theoretical work. For a given protein fold, it is probably not possible to reliably predict an "ideal" sequence. We identify and survey several aspects of the problem. Firstly, it is not clear what is the best way to score a sequence-structure pair. Secondly, there is no consensus as to what the score function should represent (free energy or some abstract measure of sequence-structure compatibility). Finally, the number of possible sequences is astronomical and searching this space poses a daunting optimisation problem. These problems are discussed in the light of recent experimental successes.

Crystalline actin tubes. V. The effect of Th4+ on actin and the role of ionic charge in tube formation.

NMR and UV spectroscopy reveal that the tetravalent thorium ion interacts with actin in an identical fashion to the trivalent lanthanide ions. UV spectra and viscometry data suggest that the actin-bound Th4+ has an ionic radius of about 110 pm. Th4+ also contributes to the formation of crystalline actin tubes under appropriate conditions. The factors governing whether tubes form appear to be cationic radius and total charge on the actin monomer, rather than the valency of the ion per se.

[Effects of arterial hyperpression on the transport and distribution of LDL and albumin in the arterial wall]. / Effets de l'hyperpression artérielle sur le transport et la distribution des LDL et de l'albumine dans la paroi artérielle.

To investigate the effect of hyperpressure on the transport of LDL and albumin in the arterial wall, we measured in vitro the uptake of both 131I-LDL and 125I-albumin in intact rabbit thoracic aorta, held at in vivo length and pressurized to 70 or 160 mmHg. Arteries were incubated for 2 h at 70 mmHg, and for 5 min, 30 min, 1 h and 2 h at 160 mmHg. The transmural distribution of the relative concentrations of LDL (CLDL) and albumin (CAlb) across the wall was determined using a serial frozen sectioning technique. At 70 mmHg, the mean medial CLDL and CAlb values were 0.0018 +/- 0.0007 and 0.0039 +/- 0.0013, respectively. At 160 mmHg, CLDL and CAlb were markedly increased. The distribution of labeled albumin was almost uniform across the media and reached a steady state after 30 min, whereas labeled LDL accumulated in the first inner layers, a steady state being achieved after 1 h. The 1-hour values of CLDL in the first and second luminal sections (0.24 +/- 0.03 and 0.13 +/- 0.05, respectively) were much higher than those of CAlb, the CLDL/CAlb ratios being 4.12 +/- 0.94 and 2.34 +/- 0.42 (p less than 0.01), respectively. In the subsequent sections, the CLDL markedly decreased and became much lower than the CAlb, the CLDL/CAlb ratio averaging 0.2 in the two thirds outer media. To investigate whether LDL was trapped at high pressure in the inner layers, vessels were exposed to a tracer-free intraluminal solution for 30 min, following a 30-minute incubation with tracers.(ABSTRACT TRUNCATED AT 250 WORDS)

Disentangling Electronic and Vibrational Coherence in the Phycocyanin-645 Light-Harvesting Complex.

Energy transfer between chromophores in photosynthesis proceeds with near-unity quantum efficiency. Understanding the precise mechanisms of these processes is made difficult by the complexity of the electronic structure and interactions with different vibrational modes. Two-dimensional spectroscopy has helped resolve some of the ambiguities and identified quantum effects that may be important for highly efficient energy transfer. Many questions remain, however, including whether the coherences observed are electronic and/or vibrational in nature and what role they play. We utilize a two-color, four-wave mixing experiment with control of the wavelength and polarization to selectively excite specific coherence pathways. For the light-harvesting complex PC645, from cryptophyte algae, we reveal and identify specific contributions from both electronic and vibrational coherences and determine an excited-state structure based on two strongly coupled electronic states and two vibrational modes. Separation of the coherence pathways also uncovers the complex evolution of these coherences and the states involved.

Coherent Vibronic Coupling in Light-Harvesting Complexes from Photosynthetic Marine Algae.

Observations of long-lived coherences in photosynthetic light-harvesting complexes utilize short pulses with broad spectral bandwidths to coherently excite multiple transitions and coherent superpositions. In order to identify the role that such quantum effects might play in efficient energy transfer, however, an alternative approach is required. We have developed a technique for two-color photon echo spectroscopy to selectively excite the pathway of interest and measure its evolution in the absence of any other excitation. We use this technique to excite a coherence pathway in phycocyanin-645 from cryptophyte algae and measure the dynamics of this coherence. A decoherence time of 500 fs was measured, and clear signatures for strong coupling between the electronic states and phonon modes were observed, allowing coherent coupling between otherwise nonresonant transitions. This provides detailed experimental evidence of the long-lived coherences and the nature of the quantum mechanical interactions between electronic states and phonon modes in phycocyanin-645 from cryptophyte marine algae.