Crystal structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein

We report the 2.1 A crystal structure of the core G protein domain of the unusual Rho family member RhoE/Rnd3 in complex with endogenous GTP and magnesium. Unlike other small G proteins, RhoE, along with two other proteins Rnd1/Rho6 and Rnd2/RhoN, does not hydrolyze GTP. The main reason for this is the presence of serines in the positions equivalent to Ala59 and Gln61 in Ras. The structure shows that there are still water molecules in similar positions to the waters thought to be involved in the hydrolysis reaction in other G proteins. The structure suggests three not necessarily exclusive explanations for the lack of hydrolysis. The lack of the conserved glutamine raises the energy of the transition state inhibiting hydrolysis. The serines may restrain the waters from moving closer to the GTP, a step that is required to attain the transition state. They also stabilize the GTP-bound conformation of switch II and could prevent conformational changes required during hydrolysis. By superposition of the RhoE structure on structures of Rho family proteins in complex with binding partners, we make predictions on RhoE interactions with these partners.     

Evidence that methylation of the FMR-I locus is responsible for variable phenotypic expression of the fragile X syndrome.

DNA at the FMR-1 locus was analyzed by Southern blot using probe StB12.3 in an unusual fragile X family with six brothers, three of whom are affected with fragile X to varying degrees, two of whom are nonpenetrant carriers, and one of whom is unaffected. Fragile X chromosome studies, detailed physical examinations, and psychological testing were completed on all six. Two of the affected brothers and the two nonpenetrant brothers were found to be methylation mosaics. The three affected males spanned the phenotypic and cognitive spectrum of the fragile X syndrome. A correlation was seen between the degree of methylation and the phenotypic expression identified in the three affected males. The two males initially classified as nonpenetrant were found to have mild phenotypic expression which consisted of minor cognitive deficits and a partial physical phenotype. These two, who were negative on fragile X chromosome studies, were found on DNA analysis to have large broad smears, with approximately 97% of the DNA unmethylated. The results described here indicate that some "nonpenetrant" carrier males may have varying amounts of methylation of the FMR-1 region, which can result in mild expression of the fragile X syndrome. The apparently mild phenotypic and cognitive expression of the fragile X syndrome in the two males, initially classified as nonpenetrant, who are mosaic for hypermethylation of an expansion of the CGG repeat in the premutation range, indicates that expression of the syndrome is not confined to males with large, hypermethylated expansions (full mutation) but has instead a gradient effect with a threshold for the full expression of the phenotype.     

Using an NMR metabolomics approach to investigate the pathogenicity of amyloid-beta and alpha-synuclein

Introduction

The pathogenicity at differing points along the aggregation pathway of many fibril-forming proteins associated with neurodegenerative diseases is unclear. Understanding the effect of different aggregation states of these proteins on cellular processes is essential to enhance understanding of diseases and provide future options for diagnosis and therapeutic intervention.

Objectives

To establish a robust method to probe the metabolic changes of neuronal cells and use it to monitor cellular response to challenge with three amyloidogenic proteins associated with neurodegenerative diseases in different aggregation states.

Method

Neuroblastoma SH-SY5Y cells were employed to design a robust routine system to perform a statistically rigorous NMR metabolomics study into cellular effects of sub-toxic levels of alpha-synuclein, amyloid-beta 40 and amyloid-beta 42 in monomeric, oligomeric and fibrillar conformations.

Results

This investigation developed a rigorous model to monitor intracellular metabolic profiles of neuronal cells through combination of existing methods. This model revealed eight key metabolites that are altered when neuroblastoma cells are challenged with proteins in different aggregation states. Metabolic pathways associated with lipid metabolism, neurotransmission and adaptation to oxidative stress and inflammation are the predominant contributors to the cellular variance and intracellular metabolite levels. The observed metabolite changes for monomer and oligomer challenge may represent cellular effort to counteract the pathogenicity of the challenge, whereas fibrillar challenge is indicative of system shutdown. This implies that although markers of stress are more prevalent under oligomeric challenge the fibrillar response suggests a more toxic environment.

Conclusion

This approach is applicable to any cell type that can be cultured in a laboratory (primary or cell line) as a method of investigating how protein challenge affects signalling pathways, providing additional understanding as to the role of protein aggregation in neurodegenerative disease initiation and progression.

     

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.     

The structure and selectivity of the SR protein SRSF2 RRM domain with RNA

SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.     

The interstitial system of the spinal trigeminal tract in the rat: anatomical evidence for morphological and functional heterogeneity

Utilizing cyto-, myelo-, and chemoarchitecture as well as connectional criteria, the present study reveals the interstitial system of the spinal trigeminal tract (InSy-SVT) in the rat to be composed of five morphologically and functionally distinct components that are distributed within spatially restricted regions of the lateral medulla. The first component is represented by scattered interstitial cells and neuropil, which extend laterally into SVT from the superficial laminae of the medullary dorsal horn (MDH). The second component, the dorsal paramarginal nucleus (PaMd), consists of a small group of marginal (lamina I)-like neurons and neuropil situated within the dorsolateral part of SVT at the rostral pole of MDH. The third component represents a trigeminal extension of the parvocellular reticular formation (V-Rpc) into the ventromedial aspect of SVT at levels extending from rostral MDH to the caudal part of trigeminal nucleus interpolaris (Vi). The fourth component, the paratrigeminal nucleus (PaV), consists of a large accumulation of neurons and neuropil situated within the dorsal part of SVT throughout the caudal half of Vi. The fifth component is the insular trigeminal-cuneatus lateralis nucleus (iV-Cul), which is a discontinuous collection of neurons and neuropil interspersed among fibers of SVT as well as wedged between it and the spinocerebellar tract. Thalamic projection neurons are located in PaMd and V-Rpc, whereas cerebellar projecting neurons are confined to iV-Cul.     

Information transmission during intra- and interspecific courtship inEphestia elutella andCadra figulilella

Using information theory, courtship posturing in the moths Ephestia elutella(Hübner) and Cadra figulilella(Gregson) was analyzed for information transmission, which was partitioned into autocovariability (intraindividual transmission) and cross-covariability (interindividual transmission). This two-factor analysis was sufficient to account for more than 60% of the behavioral variance in males of E. elutellaand in both sexes of C. figulilelladuring intraspecific courtships; however, there were large residual variances in the behavior of male and female C. figulilelladuring interspecific courtships and in E. elutellafemales during both inter- and intraspecific courtships. In E. elutella,significant levels of transmission were attributable to both inter- and intraindividual effects, whereas in C. figulilella,only autocovariability was high and no interindividual communication could be assigned to courtship postures. Although courtship in these two species was qualitatively very similar and males readily courted nonconspecific females, high levels of reproductive isolation resulted from courtship. Male C. figulilellahad 94% fewer copulations with E. elutellafemales than with conspecific females and E. elutellamales had 78% fewer copulations with C. figulilellafemales than with conspecifics. These reductions were due to a differential response in both females and males, causing inter-specific courtships to be terminated much earlier than intraspecific courtships. This discrimination indicates that interindividual communication was indeed occurring during courtship and was only partially measured by analysis of postures. Thus, communication took place largely in some other modality, most likely the chemical modality, where species specificity is suggested for both male and female pheromones.