Cross-species genetic screens identify transglutaminase 5 as a regulator of polyglutamine-expanded ataxin-1.

Many neurodegenerative disorders are caused by abnormal accumulation of misfolded proteins. In spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded (polyQ-expanded) ataxin-1 (ATXN1) causes neuronal toxicity. Lowering total ATXN1, especially the polyQ-expanded form, alleviates disease phenotypes in mice, but the molecular mechanism by which the mutant ATXN1 is specifically modulated is not understood.

Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1.

A critical question in neurodegeneration is why the accumulation of disease-driving proteins causes selective neuronal loss despite their brain-wide expression. In Spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded Ataxin-1 (ATXN1) causes selective degeneration of cerebellar and brainstem neurons. Previous studies revealed that inhibiting Msk1 reduces phosphorylation of ATXN1 at S776 as well as its levels leading to improved cerebellar function.

Losing Dnmt3a dependent methylation in inhibitory neurons impairs neural function by a mechanism impacting Rett syndrome.

Methylated cytosine is an effector of epigenetic gene regulation. In the brain, Dnmt3a is the sole 'writer' of atypical non-CpG methylation (mCH), and MeCP2 is the only known 'reader' for mCH. We asked if MeCP2 is the sole reader for Dnmt3a dependent methylation by comparing mice lacking either protein in GABAergic inhibitory neurons.

The distinct methylation landscape of maturing neurons and its role in Rett syndrome pathogenesis.

Rett syndrome (RTT) is one of the most common causes of intellectual and developmental disabilities in girls, and is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). Here we will review our current understanding of RTT, the landscape of pathogenic mutations and function of MeCP2, and culminate with recent advances elucidating the distinct DNA methylation landscape in the brain that may explain why disease symptoms are delayed and selective to the nervous system.

A Druggable Genome Screen Identifies Modifiers of α-Synuclein Levels via a Tiered Cross-Species Validation Approach.

Accumulation of α-Synuclein (α-Syn) causes Parkinson's disease (PD) as well as other synucleopathies. α-Syn is the major component of Lewy bodies and Lewy neurites, the proteinaceous aggregates that are a hallmark of sporadic PD. In familial forms of PD, mutations or copy number variations in (the α-Syn gene) result in a net increase of its protein levels.

Genome-wide distribution of linker histone H1.0 is independent of MeCP2.

Previous studies suggested that MeCP2 competes with linker histone H1, but this hypothesis has never been tested in vivo. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) of Flag-tagged-H1.0 in mouse forebrain excitatory neurons.

MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome.

Epigenetic mechanisms, such as DNA methylation, regulate transcriptional programs to afford the genome flexibility in responding to developmental and environmental cues in health and disease. A prime example involving epigenetic dysfunction is the postnatal neurodevelopmental disorder Rett syndrome (RTT), which is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). Despite decades of research, it remains unclear how MeCP2 regulates transcription or why RTT features appear 6-18 months after birth.

A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity.

Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. In this study, we address the regulatory function of this extension or 'strap' and demonstrate its responsibility for an unusual temperature dependence in ATPase rates.

Sparse labeling of proteins: structural characterization from long range constraints.

Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In these cases, sparse isotopic labeling (single or small subsets of amino acids) combined with long range paramagnetic constraints and improved computational modeling offer an alternative. This perspective provides a brief overview of this approach and two discussions of potential applications; one involving a very large system (an Hsp90 homolog) in which perdeuteration is possible and methyl-TROSY sequences can potentially be used to improve resolution, and one involving ligand placement in a glycosylated protein where resolution is achieved by single amino acid labeling (the sialyltransferase, ST6Gal1).

Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism.

While structural symmetry is a prevailing feature of homo-oligomeric proteins, asymmetry provides unique mechanistic opportunities. We present the crystal structure of full-length TRAP1, the mitochondrial Hsp90 molecular chaperone, in a catalytically active closed state. The TRAP1 homodimer adopts a distinct, asymmetric conformation, where one protomer is reconfigured via a helix swap at the middle:C-terminal domain (MD:CTD) interface.

Cross-monomer substrate contacts reposition the Hsp90 N-terminal domain and prime the chaperone activity.

The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown.

Substrate binding drives large-scale conformational changes in the Hsp90 molecular chaperone.

Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131Δ), a well-studied fragment of staphylococcal nuclease.

Conformational dynamics of the molecular chaperone Hsp90.

The ubiquitous molecular chaperone Hsp90 makes up 1-2% of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target.

Aptamer:toxin conjugates that specifically target prostate tumor cells.

We have used RNA aptamer:gelonin conjugates to target and specifically destroy cells overexpressing the known cancer biomarker prostate-specific membrane antigen (PSMA). Aptamer:toxin conjugates have an IC50 of 27 nmol/L and display an increased potency of at least 600-fold relative to cells that do not express PSMA. The aptamer not only promotes uptake into target cells but also decreases the toxicity of gelonin in non-target cells.

Labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates.

Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue.

Synthetic biology: engineering Escherichia coli to see light.

We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.