SummaryHuntingtin (Htt) is a large (348 kDa) protein, essential for embryonic development and involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and transcription regulation1,2. While an integrative understanding of Htt's biological functions is lacking, the large number of identified interactors suggests that Htt serves as a protein-protein interaction hub1,3,4. Furthermore, Huntington’s disease is caused by a mutation in the Htt gene, resulting in a pathogenic expansion of a polyglutamine (polyQ) repeat at the N-terminus of Htt5,6. However, only limited structural information on Htt is currently available. Here we employed cryo-electron microscopy (cryo-EM) to determine the structure of full-length human Htt in a complex with HAP40/F8A7 to 4 Å resolution. Htt is largely α-helical and consists of three major domains. The N- and C-terminal domains contain multiple HEAT repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat (TPR)-like organization. HAP40 binds in a cleft contacting the three Htt domains by hydrophobic and electrostatic interactions, thereby stabilizing Htt conformation. These data rationalize previous biochemical results and pave the way for an improved understanding of Htt’s diverse cellular functions.
Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Emergence and progression of HD depend on continuous expression of mutant Huntingtin protein (Htt). Therefore, blocking expression of mutant Htt might be a promising therapeutic strategy. We generated a high-capacity adenoviral (HC-Ad) vector expressing a short hairpin RNA (shRNA) targeted to exon 1 of the htt gene. In vitro, this vector efficiently inhibited Htt expression in neuronal and nonneuronal cell lines. In addition, the number of Htt-immunoreactive (IR) aggregates, a hallmark of HD pathology, was significantly reduced after gene transfer with this vector. Importantly, the attenuation of aggregate formation by shRNA was observed in vivo after stereotaxic injection into the striatum of mouse models of HD. The vector was tested in two models: the R6/2 transgenic mouse model and a mouse model based on the local injection of an adenoviral vector expressing a truncated version of mutant Htt. In both models an efficient reduction in mutant Htt aggregate load measured by decreased Htt-IR aggregate formation was observed. Our results support the further development of shRNA for HD therapy.
Camellia oil extracted from Camellia seeds is rich in unsaturated fatty acids (UFAs) and secondary metabolites beneficial to human health. However, no oil-tea tree genome has yet been published, which is a major obstacle to investigating the heredity improvement of oil-tea trees. Here, using both Illumina and PicBio sequencing technologies, we present the first chromosome-level genome sequence of the oil-tea tree species Camellia chekiangoleosa Hu. (CCH). The assembled genome consists of 15 pseudochromosomes with a genome size of 2.73 Gb and a scaffold N50 of 185.30 Mb. At least 2.16 Gb of the genome assembly consists of repetitive sequences, and the rest involves a high-confidence set of 64 608 protein-coding gene models. Comparative genomic analysis revealed that the CCH genome underwent a whole-genome duplication (WGD) event shared across the Camellia genus at ~57.48 MYA and a γ-WGT event shared across all core eudicot plants at ~120 MYA. Gene family clustering revealed that the genes involved in terpenoid biosynthesis have undergone rapid expansion. Furthermore, we determined the expression patterns of oleic acid accumulation- and terpenoid biosynthesis-associated genes in six tissues. We found that these genes tend to be highly expressed in leaves, pericarp tissues, roots, and seeds. The first chromosome-level genome of oil-tea trees will provide valuable resources for determining Camellia evolution and utilizing the germplasm of this taxon.
Heat shock transcription factors (HSFs) are central elements in the regulatory network that controls plant heat stress response. They are involved in multiple transcriptional regulatory pathways and play important roles in heat stress signaling and responses to a variety of other stresses. We identified 41 members of the HSF gene family in moso bamboo, which were distributed non-uniformly across its 19 chromosomes. Phylogenetic analysis showed that the moso bamboo HSF genes could be divided into three major subfamilies; HSFs from the same subfamily shared relatively conserved gene structures and sequences and encoded similar amino acids. All HSF genes contained HSF signature domains. Subcellular localization prediction indicated that about 80% of the HSF proteins were located in the nucleus, consistent with the results of GO enrichment analysis. A large number of stress response–associated cis-regulatory elements were identified in the HSF upstream promoter sequences. Synteny analysis indicated that the HSFs in the moso bamboo genome had greater collinearity with those of rice and maize than with those of Arabidopsis and pepper. Numerous segmental duplicates were found in the moso bamboo HSF gene family. Transcriptome data indicated that the expression of a number of PeHsfs differed in response to exogenous gibberellin (GA) and naphthalene acetic acid (NAA). A number of HSF genes were highly expressed in the panicles and in young shoots, suggesting that they may have functions in reproductive growth and the early development of rapidly-growing shoots. This study provides fundamental information on members of the bamboo HSF gene family and lays a foundation for further study of their biological functions in the regulation of plant responses to adversity.
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