Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Identification of long-lived proteins in the mitochondria reveals increased stability of the electron transport chain

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NMR Structural and Biophysical Analysis of the Disease-Linked Inner Mitochondrial Membrane Protein MPV17

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.     

Extending the Horizon of Homology Detection with Coevolution-based Structure Prediction

Traditional sequence analysis algorithms fail to identify distant homologies when they lie beyond a detection horizon. In this review, we discuss how co-evolution-based contact and distance prediction methods are pushing back this homology detection horizon, thereby yielding new functional insights and experimentally testable hypotheses. Based on correlated substitutions, these methods divine three-dimensional constraints among amino acids in protein sequences that were previously devoid of all annotated domains and repeats. The new algorithms discern hidden structure in an otherwise featureless sequence landscape. Their revelatory impact promises to be as profound as the use, by archaeologists, of ground-penetrating radar to discern long-hidden, subterranean structures. As examples of this, we describe how triplicated structures reflecting longin domains in MON1A-like proteins, or UVR-like repeats in DISC1, emerge from their predicted contact and distance maps. These methods also help to resolve structures that do not conform to a “beads-on-a-string” model of protein domains. In one such example, we describe CFAP298 whose ubiquitin-like domain was previously challenging to perceive owing to a large sequence insertion within it. More generally, the new algorithms permit an easier appreciation of domain families and folds whose evolution involved structural insertion or rearrangement. As we exemplify with α1-antitrypsin, coevolution-based predicted contacts may also yield insights into protein dynamics and conformational change. This new combination of structure prediction (using innovative co-evolution based methods) and homology inference (using more traditional sequence analysis approaches) shows great promise for bringing into view a sea of evolutionary relationships that had hitherto lain far beyond the horizon of homology detection.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Association between FABP2 Ala54Thr polymorphisms and type 2 diabetes mellitus risk: a HuGE Review and Meta‐Analysis

Many studies have examined the association between the FABP2 (rs1799883) Ala54Thr gene polymorphism and type 2 diabetes mellitus risk (T2DM) in various populations, but their results have been inconsistent. To assess this relationship more precisely, A HuGE review and meta‐analysis were performed. The PubMed and CNKI database was searched for case‐control studies published up to April 2014. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Ultimately, 13 studies, comprising 2020 T2DM cases and 2910 controls were included. Overall, for the Thr carriers (Ala/Thr and Thr/Thr) versus the wild‐type homozygotes (Ala/Ala), the pooled OR was 1.18 (95% CI = 1.04–1.34, P = 0.062 for heterogeneity), for Thr/Thr versus Ala/Ala the pooled OR was 1.17 (95% CI = 1.05–1.41 P = 0.087 for heterogeneity). In the stratified analysis by ethnicity, the significantly risks were found among Asians but not Caucasians. This meta‐analysis suggests that the FABP2 (rs1799883) Ala54Thr polymorphisms are associated with increased susceptibility to T2DM risk among Asians but not Caucasians.