The Light Chain 1 Subunit of the Microtubule-Associated Protein 1B (MAP1B) Is Responsible for Tiam1 Binding and Rac1 Activation in Neuronal Cells

Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells.     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Interplay of cis- and trans-regulatory mechanisms in the spliceosomal RNA helicase Brr2

RNA helicase Brr2 is implicated in multiple phases of pre-mRNA splicing and thus requires tight regulation. Brr2 can be auto-inhibited via a large N-terminal region folding back onto its helicase core and auto-activated by a catalytically inactive C-terminal helicase cassette. Furthermore, it can be regulated in trans by the Jab1 domain of the Prp8 protein, which can inhibit Brr2 by intermittently inserting a C-terminal tail in the enzyme's RNA-binding tunnel or activate the helicase after removal of this tail. Presently it is unclear, whether these regulatory mechanisms functionally interact and to which extent they are evolutionarily conserved. Here, we report crystal structures of Saccharomyces cerevisiae and Chaetomium thermophilum Brr2-Jab1 complexes, demonstrating that Jab1-based inhibition of Brr2 presumably takes effect in all eukaryotes but is implemented via organism-specific molecular contacts. Moreover, the structures show that Brr2 auto-inhibition can act in concert with Jab1-mediated inhibition, and suggest that the N-terminal region influences how the Jab1 C-terminal tail interacts at the RNA-binding tunnel. Systematic RNA binding and unwinding studies revealed that the N-terminal region and the Jab1 C-terminal tail specifically interfere with accommodation of double-stranded and single-stranded regions of an RNA substrate, respectively, mutually reinforcing each other. Additionally, such analyses show that regulation based on the N-terminal region requires the presence of the inactive C-terminal helicase cassette. Together, our results outline an intricate system of regulatory mechanisms, which control Brr2 activities during snRNP assembly and splicing.     

Genetic variability in Puccinia striiformis f. sp. tritici populations sampled on a local scale during natural epidemics

This study investigated genetic polymorphism on a local scale in Puccinia striiformis f. sp. tritici populations during natural epidemics, in four fields located in northern France and sampled in 1998 or 1999. Two hundred and forty-seven isolates were analyzed for their amplified fragment length polymorphism (AFLP) pattern through four primer combinations, and 194 of them were also tested for their virulence factors. Only one or two pathotypes were found in each field, and all isolates had virulence v17, matching the recently introduced Yr17 resistance gene. Polymorphism on a field scale was low. Although 67 loci were polymorphic, 77% of the isolates had the same AFLP pattern, all other patterns being rare or unique. Analyses of the genetic distance between AFLP patterns based on the Jaccard index allowed us to define 12 groups, but a bootstrap analysis showed that all isolates could be assigned to a single clonal lineage. This leads us to conclude that P. striiformis f. sp. tritici populations are clonal on a field scale in northern France.     

Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct

A protocol for the efficient isotopic labeling of large G protein‐coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L‐tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell–cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell–cell communication by the addition of indole during expression. Discrete concentrations of indole and ¹⁵N₂‐L‐tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ～15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681–1690. © 2013 Wiley Periodicals, Inc.     

Studies of 13C in the foliage reveal interactions between nutrients and water in forest fertilization experiments

The severity of a root rot disease of cereals, caused by Rhizoctonia solani Kühn AG8, was inversely correlated to the Zn status of plants in field studies in 1989 and 1990. In 1989, a preliminary survey was conducted in a farmer's field in South Australia where Zn deficiency and disease were both widespread. Zn concentration in Spear wheat plants at the 3-leaf to early tillering stage was negatively correlated with severity of the disease. For the elevent elements analysed, a correlation matrix showed that Zn had the highest, and only significant (R²=0.52^**) association with disease. The effect of Zn applications and their residual value on disease severity was further studied in a long-term field experiment in 1989 and 1990 to which Zn had been applied in 1986. There was a decrease in the area of Rhizoctonia bare patch as Zn rate was increased, a result consistent with the field survey results; the recommended rate of 2.5 kg Zn ha^–1 reduced the area affected by bare patch from 42% to 21% of the total crop area compared with no Zn application, overcame Zn deficiency and increased grain yield from 1.1 to 2.8 t ha^–1. In 1990, fresh Zn application treatments were applied to trial plots designed for this purpose, in order to compare the response with the older Zn treatments applied in 1986. The areas of bare patch in the older Zn treatments were approximately 5% greater than those in the fresh Zn treatments. The results are consistent with the hypothesis that Zn deficient plants are more susceptible to root rot caused by R. solani. Testing this hypothesis is the subject of a companion paper.     

A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers

The introduction of alkylated, nonporous poly-(styrene-divinylbenzene) microparticles in 1992 enabled the subsequent development of denaturing HPLC that has emerged as the most sensitive screening method for mutations to date. Denaturing HPLC has provided unprecedented insight into human origins and prehistoric migrations, accelerated the cloning of genes involved in mono- and polygenic traits, and facilitated the mutational analysis of more than a hundred candidate genes of human disease. A significant step toward increased sample-throughput and information content was accomplished by the recent introduction of monolithic poly(styrene-divinylbenzene) capillary columns. They have enabled the construction of capillary arrays amenable to multiplex analysis of fluorescent dye-labeled nucleic acids by laser-induced fluorescence detection. Hyphenation of denaturing HPLC with electrospray ionization mass spectrometry, on the other hand, has allowed the direct elucidation of the chemical nature of DNA variation and determination of phase of multiple alleles on a chromosome.     

Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.