Structural basis of telomeric nucleosome recognition by shelterin factor TRF1.

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1. Our structures reveal that TRF1 binds unwrapped nucleosomal DNA ends by engaging both the nucleosomal DNA and the histone octamer. Unexpectedly, TRF1 binding shifts the register of the nucleosomal DNA by 1 bp. We discovered that phosphorylation of the TRF1 C terminus and a noncanomical DNA binding surface on TRF1 are critical for its association with telomeric nucleosomes. These insights into shelterin-chromatin interactions have crucial implications for understanding telomeric chromatin organization and other roles of shelterin at telomeres including replication and transcription.

Head-to-tail polymerization by VEL proteins underpins cold-induced Polycomb silencing in flowering control.

Transcriptional silencing through the Polycomb silencing machinery utilizes a "read-write" mechanism involving histone tail modifications. However, nucleation of silencing and long-term stable transmission of the silenced state also requires P-olycomb Repressive Complex 2 (PRC2) accessory proteins, whose molecular role is poorly understood. The Arabidopsis VEL proteins are accessory proteins that interact with PRC2 to nucleate and propagate silencing at the FLOWERING LOCUS C (FLC) locus, enabling early flowering in spring. Here, we report that VEL proteins contain a domain related to an atypical four-helix bundle that engages in spontaneous concentration-dependent head-to-tail polymerization to assemble dynamic biomolecular condensates. Mutations blocking polymerization of this VEL domain prevent Polycomb silencing at FLC. Plant VEL proteins thus facilitate assembly of dynamic multivalent Polycomb complexes required for inheritance of the silenced state.

Ultrastructure and nuclear architecture of telomeric chromatin revealed by correlative light and electron microscopy.

Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.

Insights into POT1 structural dynamics revealed by cryo-EM.

Telomeres are protein-DNA complexes that protect the ends of linear eukaryotic chromosomes. Mammalian telomeric DNA consists of 5'-(TTAGGG)n-3' double-stranded repeats, followed by up to several hundred bases of a 3' single-stranded G-rich overhang. The G-rich overhang is bound by the shelterin component POT1 which interacts with TPP1, the component involved in telomerase recruitment. A previously published crystal structure of the POT1 N-terminal half bound to the high affinity telomeric ligand 5'-TTAGGGTTAG-3' showed that the first six nucleotides, TTAGGG, are bound by the OB1 fold, while the adjacent OB2 binds the last four, TTAG. Here, we report two cryo-EM structures of full-length POT1 bound by the POT1-binding domain of TPP1. The structures differ in the relative orientation of the POT1 OB1 and OB2, suggesting that these two DNA-binding OB folds take up alternative conformations. Supporting DNA binding studies using telomeric ligands in which the OB1 and OB2 binding sites were spaced apart, show that POT1 binds with similar affinities to spaced or contiguous binding sites, suggesting plasticity in DNA binding and a role for the alternative conformations observed. A likely explanation is that the structural flexibility of POT1 enhances binding to the tandemly arranged telomeric repeats and hence increases telomere protection.

A stable immature lattice packages IP6 for HIV capsid maturation.

HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

Diagnostic value of serum human epididymis protein 4 in esophageal squamous cell carcinoma.

BACKGROUND: Numerous studies have demonstrated that human epididymis protein 4 (HE4) is overexpressed in various malignant tissues including ovarian, endometrial, lung, breast, pancreatic, and gastric cancers. However, no study has examined the diagnostic impact of HE4 in patient with esophageal squamous cell carcinoma (ESCC) until now. AIM: To analyze the value of four serum tumor markers for the diagnosis of ESCC, and examine the associations of serum levels of HE4 with ESCC patients' clinicopathological characteristics. METHODS: The case group consisted of 80 ESCC patients, which were compared to a control group of 56 patients with benign esophageal disease. Serum levels of HE4, carcinoma embryonic antigen (CEA), alpha fetal protein, and carbohydrate antigen 19-9 (CA19-9) were detected by ELISA. The associations of serum HE4 levels with ESCC patients' clinicopathological characteristics such as gender, tumor location, and pathological stage were also examined after operation. RESULTS: The result of ELISA showed that serum HE4 level was significantly higher in the patients with ESCC than in the controls, and the staining intensity was inversely correlated with the pathological T and N stages. Serum HE4 levels had a sensitivity of 66.2% and specificity of 78.6% when the cutoff value was set at 3.9 ng/mL. Moreover, the combined HE4 and CA19-9 increased the sensitivity to 83.33%, and interestingly, the combination of HE4 with CEA led to the most powerful sensitivity of 87.5%. Furthermore, A positive correlation was observed between HE4 serum levels and pathological T and N stages (P = 0.0002 and 0.0017, respectively), but there was no correlation between HE4 serum levels and ESCC patient gender (P = 0.4395) or tumor location (P = 0.6777). CONCLUSION: The results of this study suggest that detection of serum HE4 levels may be useful in auxiliary diagnosis and evaluation of the progression of ESCC.

Supramolecular crosslinks enable PIC micelles with tuneable salt stability and diverse properties.

The stability of polyion complex (PIC) nanoparticles, like PIC micelles or PICsomes, in water is typically affected by added salt because salt screens the electrostatic driving force. This lack of salt stability seriously hampers numerous potential applications and a remedy is needed. Extending an earlier idea, we develop here a general strategy for preparing PIC micelles, with not only tuneable salt stability but also built-in functions. Using two different dipicolinic (DPA)-based ligands (a linear bis-ligand and a branched tris-ligand), as well as various metal ions we obtain anionic coordination polymers that subsequently co-assemble with a polycationic-neutral diblock copolymer to form PIC micelles. By a judicious choice of the metal ions and/or an appropriate mixture of the ligands we can create micellar cores with two types of reversible cross-links. In this way, we construct PIC micelles with not only tuneable and enhanced salt stability, but also tuned metal-derived properties, such as luminescence or magnetic relaxation. This non-covalent cross-link strategy, exclusively based on building block composition, is generally applicable with different metal ions and ligand combinations, and is therefore a robust approach for preparing stable and functional PIC micelles. Extension to other types of assemblies such as 'PICsomes' is possible, and therefore a range of applications becomes feasible.

Atomic-resolution map of the interactions between an amyloid inhibitor protein and amyloid ß (Aß) peptides in the monomer and protofibril states.

Self-association of amyloid ß (Aß) peptides is a hallmark of Alzheimer's disease and serves as a general prototype for amyloid formation. A key endogenous inhibitor of Aß self-association is human serum albumin (HSA), which binds ∼90% of plasma Aß. However, the exact molecular mechanism by which HSA binds Aß monomers and protofibrils is not fully understood. Here, using dark-state exchange saturation transfer NMR and relaxation experiments complemented by morphological characterization, we mapped the HSA-Aß interactions at atomic resolution by examining the effects of HSA on Aß monomers and soluble high-molecular weight oligomeric protofibrils. We found that HSA binds both monomeric and protofibrillar Aß, but the affinity of HSA for Aß monomers is lower than for Aß protofibrils (Kd values are submillimolar rather than micromolar) yet physiologically relevant because of the ∼0.6-0.7 mm plasma HSA concentration. In both Aß protofibrils and monomers, HSA targets key Aß self-recognition sites spanning the ß strands found in cross-ß protofibril structures, leading to a net switch from direct to tethered contacts between the monomeric Aß and the protofibril surface. These HSA-Aß interactions are isoform-specific, because the HSA affinity of Aß monomers is lower for Aß(1-42) than for Aß(1-40). In addition, the HSA-induced perturbations of the monomer/protofibrils pseudo-equilibrium extend to the C-terminal residues in the Aß(1-42) isoform but not in Aß(1-40). These results provide an unprecedented view of how albumin interacts with Aß and illustrate the potential of dark-state exchange saturation transfer NMR in mapping the interactions between amyloid-inhibitory proteins and amyloidogenic peptides.

Structural and Functional Insights into the Evolution and Stress Adaptation of Type II Chaperonins.

Chaperonins are essential biological complexes assisting protein folding in all kingdoms of life. Whereas homooligomeric bacterial GroEL binds hydrophobic substrates non-specifically, the heterooligomeric eukaryotic CCT binds specifically to distinct classes of substrates. Sulfolobales, which survive in a wide range of temperatures, have evolved three different chaperonin subunits (α, ß, γ) that form three distinct complexes tailored for different substrate classes at cold, normal, and elevated temperatures. The larger octadecameric ß complexes cater for substrates under heat stress, whereas smaller hexadecameric αß complexes prevail under normal conditions. The cold-shock complex contains all three subunits, consistent with greater substrate specificity. Structural analysis using crystallography and electron microscopy reveals the geometry of these complexes and shows a novel arrangement of the α and ß subunits in the hexadecamer enabling incorporation of the γ subunit.

Porphyrin-phospholipid liposomes permeabilized by near-infrared light.

The delivery of therapeutic compounds to target tissues is a central challenge in treating disease. Externally controlled drug release systems hold potential to selectively enhance localized delivery. Here we describe liposomes doped with porphyrin-phospholipid that are permeabilized directly by near-infrared light. Molecular dynamics simulations identified a novel light-absorbing monomer esterified from clinically approved components predicted and experimentally demonstrated to give rise to a more stable porphyrin bilayer. Light-induced membrane permeabilization is enabled with liposomal inclusion of 10 molar % porphyrin-phospholipid and occurs in the absence of bulk or nanoscale heating. Liposomes reseal following laser exposure and permeability is modulated by varying porphyrin-phospholipid doping, irradiation intensity or irradiation duration. Porphyrin-phospholipid liposomes demonstrate spatial control of release of entrapped gentamicin and temporal control of release of entrapped fluorophores following intratumoral injection. Following systemic administration, laser irradiation enhances deposition of actively loaded doxorubicin in mouse xenografts, enabling an effective single-treatment antitumour therapy.

Structural determinants stabilizing the axial channel of ClpP for substrate translocation.

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a ß-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.