Molecular glue CELMoD compounds are regulators of cereblon conformation.

Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.

The YΦ motif defines the structure-activity relationships of human 20S proteasome activators.

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A. A cryo-EM structure of PA26E102A-h20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation.

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome's regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

Ataxia, telangiectasia con inmunodeficiencia / Ataxia, telangiectasia with immunodeficiency

La ataxia-telangiectasia es un trastorno autosómico recesivo, caracterizado por la presencia de telangiectasias oculocutáneas, ataxia cerebelosa progresiva, inmunodeficiencia e infecciones recurrentes. Además, está relacionado con neoplasias del sistema retículo-endotelial y trastornos inmunológicos. El objetivo de la presentación es destacar el papel del dermatólogo en este tipo de trastornos neurocutáneos, así como la importancia del seguimiento a largo plazo.

Ataxia telangiectasia is an autosomal recessive disorder characterized by oculocutaneous telangiectasia, progressive cerebellar ataxia, immunodeficiency, and recurrent infections. Besides, it is related to reticuloendothelial system neoplasms and immune disorders. The aim of this presentation is to emphasize the role of the Dermatologist in this type of neurocutaneous disorders and the importance of long-term follow up.

What's the Key to Unlocking the Proteasome's Gate?

In this issue of Structure, Bolten et al. (2016) describe the organization of the mycobacterial proteasome in complex with the ATP-independent bacterial proteasome activator (Bpa, PafE). They confirm several activation motifs employed by archaea and eukaryotes and highlight differences that pose Bpa as a novel architectural class of proteasome activators.

DSF Guided Refolding As A Novel Method Of Protein Production.

The Anfinsen hypothesis, the demonstration of which led to the Nobel prize in Chemistry, posits that all information required to determine a proteins' three dimensional structure is contained within its amino acid sequence. This suggests that it should be possible, in theory, to fold any protein in vitro. In practice, however, protein production by refolding is challenging because suitable refolding conditions must be empirically determined for each protein and can be painstaking. Here we demonstrate, using a variety of proteins, that differential scanning fluorimetry (DSF) can be used to determine and optimize conditions that favor proper protein folding in a rapid and high-throughput fashion. The resulting method, which we deem DSF guided refolding (DGR), thus enables the production of aggregation-prone and disulfide-containing proteins by refolding from E. coli inclusion bodies, which would not normally be amenable to production in bacteria.

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-ß-α NDPSase fold differentiates NDPSases from ADPRases.

A redox regulatory system critical for mycobacterial survival in macrophages and biofilm development.

Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

Quistes vellosos eruptivos: un caso / Eruptive vellus cyst: a case

Los quistes vellosos eruptivos son pápulas foliculares bien delimitadas, de color piel normal, rojizas o amarillentas, que miden de 1 a 4 milímetros de diámetro y se localizan preferentemente en el tórax anterior o las extremidades. Constituyen una entidad benigna, de herencia autosómica dominante o adquirida. Se presentan habitualmente sin síntomas, aunque en ocasiones generan prurito. Se describe una niña de 7 años de edad con quistes vellosos eruptivos de presentación esporádica...(AU)

Eruptive vellus hair cysts are follicular, well-defined papules, measuring from 1 to 4 mm in diameter, normal skin color, reddish or yellowish that are preferentially located on the anterior chest or limbs. It is a benign condition, sporadic or inherited as an autosomal trait. They are clinically asymptomatic, although sometimes they may itch. We describe a 7-year-old girl that presents sporadic eruptive vellus hair cysts...(AU)

Quistes vellosos eruptivos: un caso / Eruptive vellus cyst: a case

Los quistes vellosos eruptivos son pápulas foliculares bien delimitadas, de color piel normal, rojizas o amarillentas, que miden de 1 a 4 milímetros de diámetro y se localizan preferentemente en el tórax anterior o las extremidades. Constituyen una entidad benigna, de herencia autosómica dominante o adquirida. Se presentan habitualmente sin síntomas, aunque en ocasiones generan prurito. Se describe una niña de 7 años de edad con quistes vellosos eruptivos de presentación esporádica...

Eruptive vellus hair cysts are follicular, well-defined papules, measuring from 1 to 4 mm in diameter, normal skin color, reddish or yellowish that are preferentially located on the anterior chest or limbs. It is a benign condition, sporadic or inherited as an autosomal trait. They are clinically asymptomatic, although sometimes they may itch. We describe a 7-year-old girl that presents sporadic eruptive vellus hair cysts...

[Vascular risk in outpatient from Internal Medicine departments. MICARE Study]. / Riesgo vascular en las consultas de Medicina Interna. Estudio MICARE.

BACKGROUND AND OBJECTIVES: The population attended in the Spanish Internal Medicine departments is of increasing age, but the prevalence of vascular risk factors and its degree of control are unknown, as well as the differences by type of hospital or consulting room. PATIENTS AND METHODS: Epidemiologic, transversal and metacentric study in patients ≥ 18 years treated in outpatient Internal Medicine hospital. Two-hundred and ninety physicians from 17 Autonomic Communities participated in the study. The type of hospital or consulting room was also recorded. Blood pressure control was defined as <140/90 mm Hg (<130/80 mm Hg in diabetics or patients with vascular disease), LDL-cholesterol control when<130 mg/dl (<100mg/dl in diabetic or vascular disease) and diabetes control if glycated hemoglobin was<7%. RESULTS: Data from 2,704 patients was collected (54% men) mean age (SD) 64,1 (14,5) years. Ninety-three percent of them had at least one cardiovascular risk factor: hypertension 73.9%, dyslipidemia 59.5%, abdominal obesity 43.4%, diabetes 39.5%. Fifty percent had some target organ damaged, 46.7% showed vascular disease and 71.2% a high or very high vascular risk. Control over risk factors was: hypertension 33.8%, cholesterol-LDL 40.8% and diabetes 50.7%. There were no differences between type of hospital or type of outpatient consultancy. CONCLUSIONS: Over 90% of patients treated in outpatient consultancies of Internal Medicine departments present vascular risk factors, regardless of the type of hospital or type of consulting room. Risk factors control was poor.

Foliculitis pustular eosinofílica neonatal: un caso / Eosinophilic pustular folliculitis newborn: a case

La foliculitis pustular eosinofílica (FPE) constituye una alteración folicular rara, de origen desconocido, que puede presentarse en el período neonatal. Es una enfermedad autolimitada y actualmente se la incluye dentro de las pustulosis neonatales estériles. Se comunica el caso de un niño de 8 meses de edad con episodios recurrentes de pápulas y pústulas foliculares pruriginosas en el tronco y el cuero cabelludo, presentes desde el primer día de vida. El examen de laboratorio para bacterias y hongos fue negativo. Se realizó la biopsia de una lesión, donde se observó infiltrado folicular y perifolicular constituido por eosinófilos.

Eosinophilic pustular folliculitis is an uncommon follicular disorder of unknown etiology, which may begin in the neonatal period. It is a self-limiting disease and nowadays it is considered within the sterile neonatal pustulosis. We report a case of an 8-month-old boy who had recurrent episodes of pruritic follicular papular and pustular lesions on the trunk and scalp from the first day of life. Laboratory examination looking for infections caused by bacteria and fungi were negative. A skin biopsy was performed showing both perifollicular and follicular infiltrate of eosinophils.

The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization.

The Thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence (DSF), is a fast and simple method that is based upon high-throughput measurements of protein stability. The Thermofluor method can be performed on nearly all qPCR machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations, screen for ligands or cofactors, and discover buffers and additives that maximize protein stability. This unit describes how to set up a Thermofluor method on several common models of qPCR instruments, how to prepare the samples for the assay, and how to run and analyze the resulting data. The unit also describes a 96-well screen to determine optimal buffer conditions for protein stability, which may assist in protein crystallization, and details the use of custom software, developed specifically for the analysis of data from Thermofluor screens.

Effect of continuous positive airway pressure on the risk of road accidents in sleep apnea patients.

BACKGROUND: Continuous positive airway pressure (CPAP) reduces daytime somnolence in the obstructive sleep apnea syndrome (OSAS) and may contribute to a reduction in the risk of motor vehicle accidents. OBJECTIVE: To evaluate the effects of CPAP on automobile collisions in patients with OSAS. METHODS: We compared the number of motor vehicle accidents in 80 patients with OSAS and 80 healthy subjects during the 2 years before and the 2 years after study entry, at which CPAP treatment was initiated. RESULTS: Patients with OSAS had a 2.6 times higher risk of suffering an automobile collision compared to controls (rate ratio, RR=2.57; 95% confidence interval, CI=1.30-5.05). After 2 years of CPAP treatment, the rate of collisions was reduced more than half in patients with OSAS (RR=0.41; 95% CI=0.21-0.79), but this occurred also in controls (RR=0.49; 95% CI=0.17-1.40). The magnitude of this fall between groups was not different (p for interaction=0.68), even after adjusting for body mass index, alcohol intake and Epworth scale. CONCLUSIONS: Patients with OSAS have an increased risk of suffering a traffic collision. This risk was significantly reduced after their inclusion in the study. Yet, as this reduction also occurred in the control group, this effect may not be due to CPAP therapy.

Long-term (72 hours) preservation of rat lungs.

OBJECTIVE: We sought to investigate whether the addition of ethanol to a preservation solution (as an antifreeze agent) might allow a reduction of the storage temperature to 0 degrees C without causing freezing damage and improve lung function after prolonged (72 hours) ischemia. METHODS: Lungs from Sprague-Dawley rats were ventilated and perfused ex vivo at 37 degrees C for 60 minutes in the following experimental groups: (1) the no ischemia and reperfusion (no I-R) group (n = 7), in which lungs were studied immediately after harvesting; (2) the LPD24 (n = 7) and (3) LPD72 (n = 8) groups, in which, after harvesting, lungs were flushed and immersed in low-potassium dextran solution and stored deflated at 10 degrees C for 24 and 72 hours, respectively, until reperfusion; and (4) the TEST72 group (n = 9), in which lungs were flushed and immersed in Krebs-Henseleit buffer with added ethanol (10 mL/L) after harvesting and stored deflated at 0 degrees C for 72 hours until reperfusion. RESULTS: Compared with the no I-R group, the other 3 groups had worse lung function, higher lung water content, and evidence of cell injury at reperfusion (P <.01). However, lung function at reperfusion (assessed on the basis of either effluent Po(2), peak airway pressure, or mean arterial pulmonary pressure) was better (P <.01) in the TEST72 group than in the LPD24 or LPD72 groups. Paradoxically, lung cell structure was better preserved in the LPD24 group than in the TEST72 group (or the LPD72 group). CONCLUSIONS: In this experimental model of rat lung ischemia-reperfusion injury, a low preservation temperature (0 degrees C) combined with the addition of ethanol to the preservation solution improves lung function at reperfusion after 72 hours of ischemia but fails to maintain lung cell structure.

[Prevention of vertical transmission of HIV-1 in Mallorca, Spain. Impact of antiretroviral therapy from 1995 to 2000]. / Prevención de la transmisión vertical del VIH-1 en Mallorca. Impacto de la terapia antirretroviral desde 1995 a 2000.

BACKGROUND: Our objective was to evaluate the impact of antiretroviral therapy (ART) in the prevention of maternal-fetal HIV transmission in a population of HIV-infected pregnant women. PATIENTS AND METHOD: We studied prospectively all HIV-infected pregnant women attended in our hospital from January 1995 to December 2000. We offered treatment with zidovudine (ZDV) alone or in combination according to women's requirements. RESULTS: There were 98 mother-infant pairs and we studied 93 of them. The rate of vertical transmission was 1.4% when ART was started in pregnancy. Risk of HIV transmission was greater in mothers not being treated with ART during pregnancy (relative risk [RR]: 18; 95% confidence interval [CI]: 2.2-145.4), in mothers who only received ZDV at delivery and child vs those who received ART during pregnancy (RR: 16.4; 95% CI: 1.8-145.6) and in mothers who were active intravenous drug users (RR: 9.3; 95% CI: 2.2-38.5), with significant differences between vaginal delivery and caesarean section. CONCLUSIONS: We observed a substantial benefit from ART, especially in the group of HIV-infected pregnant women who started treatment during pregnancy. Preventive interventions are needed.