Architecture of the yeast Rrp44 exosome complex suggests routes of RNA recruitment for 3' end processing

The eukaryotic core exosome (CE) is a conserved nine-subunit protein complex important for 3' end trimming and degradation of RNA. In yeast, the Rrp44 protein constitutively associates with the CE and provides the sole source of processive 3'-to-5' exoribonuclease activity. Here we present EM reconstructions of the core and Rrp44-bound exosome complexes. The two-lobed Rrp44 protein binds to the RNase PH domain side of the exosome and buttresses the bottom of the exosome-processing chamber. The Rrp44 C-terminal body part containing an RNase II-type active site is anchored to the exosome through a conserved set of interactions mainly to the Rrp45 and Rrp43 subunit, whereas the Rrp44 N-terminal head part is anchored to the Rrp41 subunit and may function as a roadblock to restrict access of RNA to the active site in the body region. The Rrp44-exosome (RE) architecture suggests an active site sequestration mechanism for strict control of 3' exoribonuclease activity in the RE complex.     

Effects of Spray and Stretch on Postneedling Soreness and Sensitivity After Dry Needling of a Latent Myofascial Trigger Point

Objectives

To investigate (1) the effect of spray and stretch versus control on reducing postneedling soreness of 1 latent myofascial trigger point (MTrP) and (2) whether higher levels of psychological distress are associated with increased postneedling pain intensity.

Design

A 72-hour follow-up, single-blind randomized controlled trial.

Setting

University community.

Participants

Healthy volunteers (N=70; 40 men, 30 women) aged 18 to 36 years (mean age, 21±4y) with latent MTrP in 1 upper trapezius muscle.

Intervention

All subjects received a dry needling application over the upper trapezius muscle. Then, participants were randomly divided into 2 groups: an intervention group, which received spray and stretch over the needled trapezius muscle, and a control group, which did not receive any intervention.

Main Outcome Measures

Visual analog scale (at postneedling, posttreatment, and 6, 12, 24, 48, and 72h after needling), pressure pain threshold (at preneedling, postneedling, and 24 and 48h after needling). Psychological distress was evaluated by using the Symptom Checklist-90-Revised.

Results

Repeated-measures analysis of variance demonstrated a significant interaction between group and time (F_3,204.8=3.19; P<.05; η_p²=.04) for changes in postneedling soreness. Between-group differences were significant only immediately after intervention (P=.002), and there were no differences found between groups after 6 hours of the intervention (P>.05). Repeated measures of covariance showed that none of the psychological covariates affected these results. Somatization, anxiety, interpersonal sensitivity, and hostility were significantly correlated (P<.05) with postneedling pain intensity. Repeated-measures analysis of variance did not show a significant effect of spray and stretch on mechanical hyperalgesia (F_2.6,175=1.9; P=.131; η_p²=.02).

Conclusions

The spray and stretch had a short-term (<6h) effect in reducing postneedling soreness of a latent MTrP. Pressure pain threshold did not significantly change after spray and stretch. Psychological factors are related to postneedling pain.     

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.     

Neurohormonal prediction of post-infarction ventricular dysfunction and coronary disease

Little information is available about the potential role of brain (type B) natriuretic peptide in patients with acute myocardial infarction. We therefore analyzed peptide levels, measured at discharge from our coronary care unit, in 56 patients admitted with a diagnosis of acute myocardial infarction. We examined peptide concentrations in the light of different features in our patients, and found a significant association between natriuretic peptide levels and the two most important prognostic factors: left ventricular ejection fraction, and the severity and extent of coronary disease. Type B natriuretic peptide was a good predictor of these features, and we conclude that concentration of type B natriuretic peptide, measured at discharge from the coronary care unit, provides important clinical and prognostic information in patients with acute myocardial infarction.     

White matter involvement on DTI-MRI in Cushing’s syndrome relates to mood disturbances and processing speed: a case-control study

Purpose

Cushing’s syndrome (CS) is an endocrine disorder due to prolonged exposure to cortisol. Recently, microstructural white matter (WM) alterations detected by diffusion tensor imaging (DTI) have been reported in CS patients, and related to depression, but other functional significances. remain otherwise unclear. We aimed at investigating in more depth mood symptoms in CS patients, and how these relate to cognition (information processing speed), and to WM alterations on DTI.

Methods

The sample comprised 35 CS patients and 35 healthy controls. Beck Depression Inventory-II (BDI-II) was used to measure depressive symptoms, State-Trait Anxiety Inventory (STAI) to assess anxiety, and processing speed was measured by the Symbol Digit Modalities Test (SDMT). DTI studies were acquired using a 3-Tesla Philips-Achieva MR-facility. Voxelwise statistical analysis of fractional anisotropy (FA), mean, axial and radial diffusivities (MD, AD, RD) data were performed using FMRIB Software Library. Correlation analysis were obtained between mood and processing speed variables, and FA, MD, AD and RD values, taking both CS patients and healthy controls.

Results

Active, controlled and cured CS patients showed greater depression (F?=?12.4, p?<?0.001), anxious state (F?=?4.8, p?=?0.005) and anxious trait (F?=?9.6, p?<?0.001) scores, than controls. Using the entire sample, depression scores correlated negatively to FA and positively to RD values. Although there were no differences in processing speed between groups, SDMT scores correlated positively to both FA and AD values.

Conclusions

There were greater depressive and anxious symptoms in CS patients than in healthy controls, but no difference in processing speed. However, DTI is related to depression and information processing speed in CS.

     

Prevalence of cardiac amyloidosis among elderly patients with systolic heart failure or conduction disorders

Objective: Cardiac amyloid infiltration can lead to systolic heart failure (HF) or to conduction disorders (CD). Patients with transthyretin (ATTR) amyloidosis are particularly exposed. We sought to determine the prevalence of ATTR and AL among patients >60?years admitted with CD or unexplained systolic HF and increased wall thickness.

Materials and Methods: We studied 143 patients (57% males, 79?±?9?years) with HF (N?=?28) or CD requiring pacemaker implantation (N?=?115). In total, 139 (97%) patients (28 with HF and 111 with CD) underwent ^99mTc-DPD scintigraphy to detect ATTR, and 105 (73%; 19 HF and 86?CD) underwent AL screening.

Results: Five patients (4%; 95%CI:0–7%) exhibited wild-type ATTR (ATTRwt) amyloidosis, 2 (2%; 95%CI:0–4%) had CD and 3 (11%; 95%CI:0–23%) HF. No patient showed AL. The 2 ATTRwt patients with CD were previously asymptomatic, did not show classical ECG signs and exhibited mild LV hypertrophy with preserved LVEF. By contrast, all ATTRwt patients with HF had ECG and echocardiographic signs of amyloid. During a mean follow-up of 18?±?11?months, 3(60%) patients with ATTRwt amyloidosis (1?CD and 2 HF) and 14(10.4%) without died.

Conclusion: Prevalence of ATTRwt amyloidosis in patients with CD requiring pacemaker is low. Although, additional studies are needed, prevalence seems to be higher in elderly patients with systolic HF.     

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species

α-synuclein aggregation is present in Parkinson’s disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Aberrant aggregation of α-synuclein (α-syn) into amyloid fibrils and subsequent accumulation into intracellular inclusions is a hallmark of neurodegenerative disorders such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy (1–3). In these diseases, soluble α-syn monomers misfold and self-assemble, forming small oligomeric species that retain the highly disordered structure of the monomeric state (4). These species are rather unstable and can undergo structural rearrangements, including a gain in β-sheet structure that generates more stable species (4, 5). β-structured oligomers can grow further through monomer addition or self-association, finally giving rise to well-defined amyloid fibrils (4–6). Despite the controversial evidence about the relationship between the different species that populate the aggregation process and cellular toxicity, the prevalent view is that both intermediate oligomers and small fibrils are neurotoxic (7). Due to their abnormal interactions with cellular components, certain types of oligomers are key pathogenic agents in the development of the disease (8–10). In particular, they can disrupt membranes, induce oxidative stress, dysregulate calcium homeostasis, cause mitochondria dysfunction, or impair the proteasome system (11). Furthermore, α-syn oligomers have been implicated in the spreading of the disease, as these aggregates can be transmitted between cells (12, 13). Small fibrils have also been related to intercellular spreading and propagation of neurodegeneration (14–18). In contrast, large amyloid aggregates are believed to be relatively inert, as their highly ordered packing and slow diffusion reduces undesired interactions with cellular components. Even so, large aggregates can generate intermediate species that contribute to cytotoxicity through secondary processes such as fragmentation or nucleation on the aggregate surface (19, 20).To counteract the toxic effect of protein aggregates, cells have evolved a sophisticated protein homeostasis network that coordinates protein synthesis, folding, disaggregation and degradation (21). This network is composed of the translational machinery, molecular chaperones and cochaperones, the ubiquitin-proteasome system, and the autophagy machinery. The way this network tackles amyloid aggregates remains poorly understood. It has been previously reported that the constitutive human Hsp70 (Hsc70) in collaboration with its Hsp40 cochaperone (Hdj1 or DnaJB1) slowly disassembles preformed α-syn fibrils (22). This activity was further stimulated by adding the NEF Hsp110 (Apg2). HspB5, a small heat shock protein also known as αB-crystallin, potentiated α-syn fibril disassembly by the ternary chaperone mixture. Although this chaperone combination was able to disaggregate fibrils, they did it in a timescale of weeks through a depolymerization process. Only when Hsp104, a yeast representative of the Hsp100 family able of fragmenting fibrils, was added to the mixture, disassembly occurred within hours (22). The lack of Hsp104 homologs in metazoans questioned whether this activity was physiologically relevant in humans. A later study revealed that a chaperone complex composed solely of members of the Hsp70, Hsp40, and Hsp110 families was able to efficiently reverse α-syn amyloid fibrils through both fragmentation and depolymerization, generating smaller fibrils, oligomers, and, ultimately, monomers (23). Despite the importance of this emerging disaggregase functionality, its mechanism of action remains largely unknown. Recently, the same chaperone mixture has been reported to also disaggregate tau and Htt fibrils (24–26), pointing to this Hsp70-based machinery as a potential human amyloid disaggregase.The two-fold aim of this work is, firstly, to test whether human disaggregase remodels with the same efficiency the different aggregates that populate the complex process of amyloid formation and, secondly, to shed light on the key mechanisms involved in the disassembly of amyloids. We show that the human disaggregase system disassembles toxic oligomers and short fibrils much better than large, less toxic fibrils, and that it does so by an enhanced destabilization of the small aggregated forms. Explicitly, fibril disassembly involves destabilization of the fibril ends and unzipping of the protofilaments, which allow depolymerization. The fast propagation of protofilament depolymerization toward the opposite fibril end is consistent with entropic pulling forces exerted by Hsc70 upon binding the fibril surface.     

The binding conformation of Taxol in beta-tubulin: a model based on electron crystallographic density

The chemotherapeutic drug Taxol is known to interact within a specific site on beta-tubulin. Although the general location of the site has been defined by photoaffinity labeling and electron crystallography, the original data were insufficient to make an absolute determination of the bound conformation. We have now correlated the crystallographic density with analysis of Taxol conformations and have found the unique solution to be a T-shaped Taxol structure. This T-shaped or butterfly structure is optimized within the beta-tubulin site and exhibits functional similarity to a portion of the B9-B10 loop in the alpha-tubulin subunit. The model provides structural rationalization for a sizeable body of Taxol structure-activity relationship data, including binding affinity, photoaffinity labeling, and acquired mutation in human cancer cells.