Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast.

Intracellular triacylglycerol (TAG) is a ubiquitous energy storage lipid also involved in lipid homeostasis and signaling. Comparatively, little is known about TAG's role in other cellular functions. Here we show a pro-longevity function of TAG in the budding yeast Saccharomyces cerevisiae. In yeast strains derived from natural and laboratory environments a correlation between high levels of TAG and longer chronological lifespan was observed. Increased TAG abundance through the deletion of TAG lipases prolonged chronological lifespan of laboratory strains, while diminishing TAG biosynthesis shortened lifespan without apparently affecting vegetative growth. TAG-mediated lifespan extension was independent of several other known stress response factors involved in chronological aging. Because both lifespan regulation and TAG metabolism are conserved, this cellular pro-longevity function of TAG may extend to other organisms.

Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3ß and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1, and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter cell lines and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation - suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation elongation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

Yeast Tripartite Biosensors Sensitive to Protein Stability and Aggregation Propensity.

In contrast to the myriad approaches available to study protein misfolding and aggregation in vitro, relatively few tools are available for the study of these processes in the cellular context. This is in part due to the complexity of the cellular environment which, for instance, interferes with many spectroscopic approaches. Here, we describe a tripartite fusion approach that can be used to assess in vivo protein stability and solubility in the cytosol of Saccharomyces cerevisiae. Our biosensors contain tripartite fusions in which a protein of interest is inserted into antibiotic resistance markers. These fusions act to directly link the aggregation susceptibility and stability of the inserted protein to antibiotic resistance. We demonstrate a linear relationship between the thermodynamic stabilities of variants of the model folding protein immunity protein 7 (Im7) fused into the resistance markers and their antibiotic resistance readouts. We also use this system to investigate the in vivo properties of the yeast prion proteins Sup35 and Rnq1 and proteins whose aggregation is associated with some of the most prevalent neurodegenerative misfolding disorders, including peptide amyloid beta 1-42 (Aß42), which is involved in Alzheimer's disease, and protein α-synuclein, which is linked to Parkinson's disease.

Hyperphosphorylated tau aggregation and cytotoxicity modulators screen identified prescription drugs linked to Alzheimer's disease and cognitive functions.

The neurodegenerative Alzheimer's disease (AD) affects more than 30 million people worldwide. There is thus far no cure or prevention for AD. Aggregation of hyperphosphorylated tau in the brain correlates with the cognitive decline of patients of AD and other neurodegenerative tauopathies. Intracerebral injection of tau aggregates isolated from tauopathy brains causes similar pathology in the recipient mice, demonstrating the pathogenic role of abnormally phosphorylated tau. Compounds controlling the aggregation of hyperphosphorylated tau therefore are probable modulators for the disease. Here we report the use of recombinant hyperphosphorylated tau (p-tau) to identify potential tauopathy therapeutics and risk factors. Hyperphosphorylation renders tau prone to aggregate and to impair cell viability. Taking advantage of these two characters of p-tau, we performed a screen of a 1280-compound library, and tested a selective group of prescription drugs in p-tau aggregation and cytotoxicity assays. R-(-)-apomorphine and raloxifene were found to be p-tau aggregation inhibitors that protected p-tau-treated cells. In contrast, a subset of benzodiazepines exacerbated p-tau cytotoxicity apparently via enhancing p-tau aggregation. R-(-)apomorphine and raloxifene have been shown to improve cognition in animals or in humans, whereas benzodiazepines were linked to increased risks of dementia. Our results demonstrate the feasibility and potential of using hyperphosphorylated tau-based assays for AD drug discovery and risk factor identification.

Hyperphosphorylation Renders Tau Prone to Aggregate and to Cause Cell Death.

Alzheimer's disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer's disease mechanistic and drug discovery studies.

Tripartite Chromatin Localization of Budding Yeast Shugoshin Involves Higher-Ordered Architecture of Mitotic Chromosomes.

The spindle assembly checkpoint (SAC) is key to faithful segregation of chromosomes. One requirement that satisfies SAC is appropriate tension between sister chromatids at the metaphase-anaphase juncture. Proper tension generated by poleward pulling of mitotic spindles signals biorientation of the underlying chromosome. In the budding yeast, the tension status is monitored by the conserved Shugoshin protein, Sgo1p, and the tension sensing motif (TSM) of histone H3. ChIP-seq reveals a unique TSM-dependent, tripartite domain of Sgo1p in each mitotic chromosome. This domain consists of one centromeric and two flanking peaks 3 - 4 kb away, present exclusively in mitosis. Strikingly, this trident motif coincides with cohesin localization, but only at the centromere and the two immediate adjacent loci, despite that cohesin is enriched at numerous regions throughout mitotic chromosomes. Chromosome conformation capture assays reveal apparent looping at the centromeric and pericentric regions. The TSM-Sgo1p-cohesin triad is therefore at the center stage of higher-ordered chromatin architecture for error-free segregation.

A Failsafe for Sensing Chromatid Tension in Mitosis with the Histone H3 Tail in Saccharomyces cerevisiae.

Mitotic fidelity is ensured by achieving biorientation on all paired chromosomes. The key signal for proper chromosome alignment is the tension between sister chromatids created by opposing poleward force from the spindles. In the budding yeast, the tension-sensing function requires that the Shugoshin protein, Shugoshin 1, be recruited to the centromeres and the neighboring pericentric regions. Concerted actions integrating proteins at centromeres and pericentromeres create highly specific Shugoshin 1 domains on mitotic chromosomes. We have previously reported that an important regulatory region on histone H3, termed the tension-sensing motif (TSM), is responsible for retaining Shugoshin 1 at pericentromeres. The TSM is negatively regulated by the acetyltransferase Gcn5p, but the underlying mechanism was elusive. In this work, we provide evidence that, when the TSM function is impaired, the histone H3 tail adopts a role that complements the damaged TSM to ensure faithful mitosis. This novel function of the H3 tail is controlled by Gcn5p, which targets selective lysine residues. Mutations to K14 and K23 ameliorate the mitotic defects resulting from TSM mutations. The restoration of faithful segregation is accompanied by regaining Shugoshin 1 access to the pericentric regions. Our data reveal a novel pathway for mitotic Shugoshin 1 recruitment and further reinforce the active role played by chromatins during their segregation in mitosis.

Identification of Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae and Its Regulation by Histone Modifying Enzymes.

To ensure genome stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. In budding yeast, the residue Gly44 of histone H3 is critical for retaining the conserved Shugoshin protein Sgo1p at the pericentromeres for monitoring the tension status during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form the core of a tension sensing motif (TSM). Similar to the previously reported G44S mutant, K42A, G44A, and T45A alleles all rendered cells unable to respond to erroneous spindle attachment, a phenotype suppressed by Sgo1p overexpression. TSM functions by physically recruiting or retaining Sgo1p at pericentromeres as evidenced by chromatin immunoprecipitation and by in vitro pulldown experiments. Intriguingly, the function of TSM is likely regulated by multiple histone modifying enzymes, including the histone acetyltransferase Gcn5p, and deacetylases Rpd3p and Hos2p Defects caused by TSM mutations can be suppressed by the expression of a catalytically inactive mutant of Gcn5p Conversely, G44S mutant cells exhibit prominent chromatin instability phenotype in the absence of RPD3 Importantly, the gcn5- suppressor restores the tension sensing function in tsm- background in a fashion that bypasses the need of stably associating Sgo1p with chromatin. These results demonstrate that the TSM of histone H3 is a key component of a mechanism that ensures faithful segregation, and that interaction with chromatin modifying enzymes may be an important part of the mitotic quality control process.

Resolving acetylated and phosphorylated proteins by neutral urea Triton-polyacrylamide gel electrophoresis: NUT-PAGE.

Protein acetylation and phosphorylation are key modifications that regulate both normal and pathological protein functions. The gel systems currently used for analyzing modified proteins require either expensive reagents or time-consuming second dimension electrophoresis. Here we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. The neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system separates proteins based on their charge at pH 7.0 and generates discrete bands from each acetylated and/or phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents and requires only a single dimension of electrophoresis. We demonstrate the effectiveness of this system by analyzing the phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative for resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge.

Structural insights into the classification of Mud Crab Reovirus.

Cryo-electron microscopy was applied to analyze mud crab reovirus (MCRV), which causes 'sleeping disease' in mud crab, Scylla serrata, a marine species cultured in China. We present here the three dimensional structure of MCRV at 13.8Å resolution. The outer capsid shell is composed of 260 trimers with complete T=13 icosahedral symmetry. A major difference between MCRV and previously reported aquareoviruses is that it lacks a pentameric turret structure. These results together with recently published molecular biological evidence (Deng et al., 2012) indicate that, from a structural perspective, MCRV should be classified as a new member of the family Reoviridae.

Sequence analysis of 12 genome segments of mud crab reovirus (MCRV).

Mud crab reovirus (MCRV) is the causative agent of a serious disease with high mortality in cultured mud crab (Scylla serrata). This study sequenced and analyzed 12 genome segments of MCRV. The 12 genome segments had a total length of 24.464 kb, showing a total G+C content of 41.29% and predicted 15 ORFs. Sequence analysis showed that the majority of MCRV genes shared low homology with the counterpart genes of other reoviruses, e.g., the amino acid identity of RNA-dependent RNA polymerase (RdRp) was lower than 13.0% compared to the RdRp sequences of other reoviruses. Nucleotide and amino acid sequences of RdRp and capping enzyme suggested MCRV as a single group. Further genome-based phylogenetical analysis of conserved termini and reovirus polymerase motif indicates that this MCRV belongs to a new genus of the Reoviridae family, tentatively named as Crabreovirus.

Identification of the structural proteins of VP1 and VP2 of a novel mud crab dicistrovirus.

Mud crab dicistrovirus (MCDV), a newly identified single-stranded positive RNA virus, is an important pathogen that causes serious economic losses to mud crab aquaculture. In this study, MCDV was purified, and three structural proteins of MCDV were separated by SDS-PAGE. The N-terminal 15 amino acids were sequenced and aligned with the main structural proteins of other dicistrovirus. The three structural proteins were named VP1, VP2 and VP3. Monoclonal antibodies (MAbs) against the two main structural proteins, VP1 and VP2, were prepared, and the two structural proteins were then identified using these MAbs. The results of Western blot analyses demonstrated that five MAbs recognised VP1 and two recognised VP2. The results of immunogold transmission electron microscopy (IEM) revealed that the epitopes of the two structural proteins recognised by the MAbs were located at the outer surface of the virions, which suggested that the two structural proteins are MCDV capsid proteins. The identification of the two structural proteins of MCDV is useful for studying their functions, as well as the mechanism of infection and the pathogenesis of MCDV.

Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key signaling adaptor protein not only for the TNFR superfamily but also for the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. To investigate TRAF6 function in invertebrate innate immune responses, Litopenaeus vannamei TRAF6 (LvTRAF6) was identified and characterized. The full-length cDNA of LvTRAF6 is 2823bp long, with an open reading frame (ORF) encoding a putative protein of 594 amino acids, including a RING-type Zinc finger, two TRAF-type Zinc fingers, a coiled-coil region, and a meprin and TRAF homology (MATH) domain. The overall amino acid sequence identity between LvTRAF6 and other known TRAF6s is 22.2-33.3%. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvTRAF6 could activate the promoters of antimicrobial peptide genes (AMPs), including Drosophila Attacin A and Drosomycin, and shrimp Penaeidins. Real-time quantitative PCR (qPCR) indicated that LvTRAF6 was constitutively expressed in various tissues of L. vannamei. After Vibrio alginolyticus and white spot syndrome virus (WSSV) challenge, LvTRAF6 was down-regulated, though with different expression patterns in the intestine compared to other tissues. After WSSV challenge, LvTRAF6 was up-regulated 2.7- and 2.3-fold over the control at 3h in gills and hepatopancreas, respectively. These results indicated that LvTRAF6 may play a crucial role in antibacterial and antiviral responses via regulation of AMP gene expression.

An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes.

Invertebrates rely on innate immunity as the first line defense against microbes. In Drosophila, the inducible antimicrobial peptides (AMPs) regulated by the Toll and immune deficiency (Imd) pathways are important effectors in innate immunity. Here we report an immune deficiency homolog (LvIMD) from the white shrimp, Litopenaeus vannamei. The full-length cDNA of LvIMD is 758 bp with an open reading frame of 483 bp that encodes a putative protein of 160 amino acids including a death domain at the C-terminus. LvIMD death domain shows similarity to that of Drosophila IMD and human receptor interacting protein 1 (RIP1) of the tumor necrosis factor receptor (TNFR) pathway, with 27.9% and 26.4% identity, respectively. Phylogenetic analysis shows that LvIMD clusters with a predicted protein from the starlet sea anemone (Nematostella vectensis) independent to insect IMDs and vertebrates RIP1s. LvIMD mRNA is expressed in most tissues and is induced in hepatopancreas and hemocytes after immune challenge. Luciferase reporter assays confirm that LvIMD is able to induce the expression of AMP genes, including Drosophila Attacin A and shrimp Penaeidin 4 in S2 cells. To our knowledge, this is the first report that LvIMD participates in innate signaling to activate the expression of AMP genes in shrimp.