A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly ...characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module’s molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.
Meta-analysis and meta-regression were used to evaluate whether evidence to date demonstrates deficits in procedural memory in individuals with specific language impairment (SLI), and to examine ...reasons for inconsistencies of findings across studies. The Procedural Deficit Hypothesis (PDH) proposes that SLI is largely explained by abnormal functioning of the frontal-basal ganglia circuits that support procedural memory. It has also been suggested that declarative memory can compensate for at least some of the problems observed in individuals with SLI. A number of studies have used Serial Reaction Time (SRT) tasks to investigate procedural learning in SLI. In this report, results from eight studies that collectively examined 186 participants with SLI and 203 typically-developing peers were submitted to a meta-analysis. The average mean effect size was .328 (CI95: .071, .584) and was significant. This suggests SLI is associated with impairments of procedural learning as measured by the SRT task. Differences among individual study effect sizes, examined with meta-regression, indicated that smaller effect sizes were found in studies with older participants, and in studies that had a larger number of trials on the SRT task. The contributions of age and SRT task characteristics to learning are discussed with respect to impaired and compensatory neural mechanisms in SLI.
Current small-molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket (SII-P), exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the ...GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.
•New advances have made it possible to determine structure of complexes containing ubiquitinated nucleosomes.•X-ray crystallographic and EM studies shed light on how the SAGA complex engages ...nucleosomes containing monoubiquitinated H2B.•The intact SAGA complex adopts multiple discrete conformations.•An EM study shows how 53BP1 recognizes tandem ubiquitin and methyl marks in the nucleosome.
Histone ubiquitination plays a non-degradative role in regulating transcription and the DNA damage response. A mechanistic understanding of this chromatin modification has lagged that of small histone modifications because of the technical challenges in preparing ubiquitinated nucleosomes. The recent structure of the DUB module of the SAGA coactivator complex bound to a nucleosome containing monoubiquitinated H2B has provided the first view of how specialized subunits target this enzyme to its substrate. Single particle electron microscopy of the intact SAGA coactivator suggests how the DUB module and histone acetyltransferase module engage a nucleosomal substrate. A cryo EM study of 53BP1 bound to nucleosomes containing ubiquitinated H2A and H4 methylated at K20 extends our understanding of recognition of biologically distinct combinations of chromatin marks through multivalent interactions.
Although learning a second language (L2) as an adult is notoriously difficult, research has shown that adults can indeed attain native language-like brain processing and high proficiency levels. ...However, it is important to then retain what has been attained, even in the absence of continued exposure to the L2--particularly since periods of minimal or no L2 exposure are common. This event-related potential (ERP) study of an artificial language tested performance and neural processing following a substantial period of no exposure. Adults learned to speak and comprehend the artificial language to high proficiency with either explicit, classroom-like, or implicit, immersion-like training, and then underwent several months of no exposure to the language. Surprisingly, proficiency did not decrease during this delay. Instead, it remained unchanged, and there was an increase in native-like neural processing of syntax, as evidenced by several ERP changes--including earlier, more reliable, and more left-lateralized anterior negativities, and more robust P600s, in response to word-order violations. Moreover, both the explicitly and implicitly trained groups showed increased native-like ERP patterns over the delay, indicating that such changes can hold independently of L2 training type. The results demonstrate that substantial periods with no L2 exposure are not necessarily detrimental. Rather, benefits may ensue from such periods of time even when there is no L2 exposure. Interestingly, both before and after the delay the implicitly trained group showed more native-like processing than the explicitly trained group, indicating that type of training also affects the attainment of native-like processing in the brain. Overall, the findings may be largely explained by a combination of forgetting and consolidation in declarative and procedural memory, on which L2 grammar learning appears to depend. The study has a range of implications, and suggests a research program with potentially important consequences for second language acquisition and related fields.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular ...pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.
Prostate cancer that progresses after enzalutamide treatment is poorly responsive to further antiandrogen therapy, and paradoxically, rapid cycling between high and low serum testosterone ...concentrations (bipolar androgen therapy BAT) in this setting might induce tumour responses. We aimed to evaluate BAT in patients with metastatic castration-resistant prostate cancer that progressed after enzalutamide.
We did this single-centre, open-label, phase 2, multicohort study in the USA. We included patients aged 18 years or older who had histologically confirmed and radiographically documented metastatic castration-resistant prostate cancer, with no more than two previous second-line hormonal therapies, and a castrate concentration of testosterone. Patients were asymptomatic, with Eastern Cooperative Oncology Group performance status of 0–2, and did not have high-risk lesions for tumour flare (eg, >5 sites of visceral disease or bone lesions with impending fracture). For the cohort reported here, we required patients to have had progression on enzalutamide with a continued prostate-specific antigen (PSA) rise after enzalutamide treatment discontinuation. Patients received BAT, which consisted of intramuscular testosterone cipionate 400 mg every 28 days until progression and continued luteinising hormone-releasing hormone agonist therapy. Upon progression after BAT, men were rechallenged with oral enzalutamide 160 mg daily. The co-primary endpoints were investigator-assessed 50% decline in PSA concentration from baseline (PSA50) for BAT (for all patients who received at least one dose) and for enzalutamide rechallenge (based on intention-to-treat analysis). These data represent the final analysis for the post-enzalutamide cohort, while two additional cohorts (post-abiraterone and newly castration-resistant prostate cancer) are ongoing. The trial is registered with ClinicalTrials.gov, number NCT02090114.
Between Aug 28, 2014, and May 18, 2016, we accrued 30 eligible patients and treated them with BAT. Nine (30%; 95% CI 15–49; p<0·0001) of 30 patients achieved a PSA50 to BAT. 29 patients completed BAT and 21 proceeded to enzalutamide rechallenge, of whom 15 (52%; 95% CI 33–71; p<0·0001) achieved a PSA50 response. During BAT, the only grade 3–4 adverse event occurring in more than one patient was hypertension (three 10% patients). Other grade 3 or worse adverse events occurring during BAT in one 3% patient each were pulmonary embolism, myocardial infarction, urinary obstruction, gallstone, and sepsis. During enzalutamide retreatment, no grade 3–4 toxicities occurred in more than one patient. No treatment-related deaths were reported during either BAT or enzalutamide retreatment.
BAT is a safe therapy that resulted in responses in asymptomatic men with metastatic castration-resistant prostate cancer and also resensitisation to enzalutamide in most patients undergoing rechallenge. Further studies with BAT are needed to define the potential clinical role for BAT in the management of metastatic castration-resistant prostate cancer and the optimal strategy for sequencing between androgen and antiandrogen therapies in metastatic castration-resistant prostate cancer to maximise therapeutic benefit to patients.
National Institutes of Health and National Cancer Institute.
MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in ...mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 “seed matches” and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.
The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of ...feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.
The endoplasmic reticulum (ER) is the primary organelle for folding and maturation of secretory and transmembrane proteins. Inability to meet protein-folding demand leads to "ER stress" and activates ...IRE1α, an ER transmembrane kinase-endoribonuclease (RNase). IRE1α promotes adaptation through splicing Xbp1 mRNA or apoptosis through incompletely understood mechanisms. Here, we found that sustained IRE1α RNase activation caused rapid decay of select microRNAs (miRs -17, -34a, -96, and -125b) that normally repress translation of Caspase-2 mRNA, and thus sharply elevates protein levels of this initiator protease of the mitochondrial apoptotic pathway. In cell-free systems, recombinant IRE1α endonucleolytically cleaved microRNA precursors at sites distinct from DICER. Thus, IRE1α regulates translation of a proapoptotic protein through terminating microRNA biogenesis, and noncoding RNAs are part of the ER stress response.
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