Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor ...(TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.
Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. ...These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.
Motility helps many pathogens swim through the highly viscous intestinal mucus. Given the differing outcomes of Campylobacter concisus infection, the motility of eight C. concisus strains isolated ...from patients with Crohn's disease (n=3), acute (n=3) and chronic (n=1) gastroenteritis and a healthy control (n=1) were compared. Following growth on solid or liquid media the eight strains formed two groups; however, the type of growth medium did not affect motility. In contrast, following growth in viscous liquid medium seven of the eight strains demonstrated significantly decreased motility. In media of increasing viscosities the motility of C. concisus UNSWCD had two marked increases at viscosities of 20.0 and 74.7 centipoises. Determination of the ability of UNSWCD to swim through a viscous medium, adhere to and invade intestinal epithelial cells showed that while adherence levels significantly decreased with increasing viscosity, invasion levels did not significantly change. In contrast, adherence to and invasion of UNSWCD to mucus-producing intestinal cells increased upon accumulation of mucus, as did bacterial aggregation. Given this aggregation, we determined the ability of the eight C. concisus strains to form biofilms, and showed that all strains formed biofilms. In conclusion, the finding that C. concisus strains could be differentiated into two groups based on their motility may suggest that strains with high motility have an increased ability to swim through the intestinal mucus and reach the epithelial layer.
NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to ...NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein sample that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform sampling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.
MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL ...revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a β-strand and repositioning of the functionally important “BB-loop” region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.
Molecular Minimal Residual Disease in Acute Myeloid Leukemia Jongen-Lavrencic, Mojca; Grob, Tim; Hanekamp, Diana ...
New England journal of medicine/The New England journal of medicine,
2018-Mar-29, Letnik:
378, Številka:
13
Journal Article
Recenzirano
Odprti dostop
Patients with acute myeloid leukemia (AML) often reach complete remission, but relapse rates remain high. Next-generation sequencing enables the detection of molecular minimal residual disease in ...virtually every patient, but its clinical value for the prediction of relapse has yet to be established.
We conducted a study involving patients 18 to 65 years of age who had newly diagnosed AML. Targeted next-generation sequencing was carried out at diagnosis and after induction therapy (during complete remission). End points were 4-year rates of relapse, relapse-free survival, and overall survival.
At least one mutation was detected in 430 out of 482 patients (89.2%). Mutations persisted in 51.4% of those patients during complete remission and were present at various allele frequencies (range, 0.02 to 47%). The detection of persistent DTA mutations (i.e., mutations in DNMT3A, TET2, and ASXL1), which are often present in persons with age-related clonal hematopoiesis, was not correlated with an increased relapse rate. After the exclusion of persistent DTA mutations, the detection of molecular minimal residual disease was associated with a significantly higher relapse rate than no detection (55.4% vs. 31.9%; hazard ratio, 2.14; P<0.001), as well as with lower rates of relapse-free survival (36.6% vs. 58.1%; hazard ratio for relapse or death, 1.92; P<0.001) and overall survival (41.9% vs. 66.1%; hazard ratio for death, 2.06; P<0.001). Multivariate analysis confirmed that the persistence of non-DTA mutations during complete remission conferred significant independent prognostic value with respect to the rates of relapse (hazard ratio, 1.89; P<0.001), relapse-free survival (hazard ratio for relapse or death, 1.64; P=0.001), and overall survival (hazard ratio for death, 1.64; P=0.003). A comparison of sequencing with flow cytometry for the detection of residual disease showed that sequencing had significant additive prognostic value.
Among patients with AML, the detection of molecular minimal residual disease during complete remission had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent mutations that are associated with clonal hematopoiesis did not have such prognostic value within a 4-year time frame. (Funded by the Queen Wilhelmina Fund Foundation of the Dutch Cancer Society and others.).
Substantial heterogeneity within mutant TP53 acute myeloid leukemia (AML) and myelodysplastic syndrome with excess of blast (MDS-EB) precludes the exact assessment of prognostic impact for individual ...patients. We performed in-depth clinical and molecular analysis of mutant TP53 AML and MDS-EB to dissect the molecular characteristics in detail and determine its impact on survival. We performed next-generation sequencing on 2200 AML/MDS-EB specimens and assessed the TP53 mutant allelic status (mono- or bi-allelic), the number of TP53 mutations, mutant TP53 clone size, concurrent mutations, cytogenetics, and mutant TP53 molecular minimal residual disease and studied the associations of these characteristics with overall survival. TP53 mutations were detected in 230 (10.5%) patients with AML/MDS-EB with a median variant allele frequency of 47%. Bi-allelic mutant TP53 status was observed in 174 (76%) patients. Multiple TP53 mutations were found in 49 (21%) patients. Concurrent mutations were detected in 113 (49%) patients. No significant difference in any of the aforementioned molecular characteristics of mutant TP53 was detected between AML and MDS-EB. Patients with mutant TP53 have a poor outcome (2-year overall survival, 12.8%); however, no survival difference between AML and MDS-EB was observed. Importantly, none of the molecular characteristics were significantly associated with survival in mutant TP53 AML/MDS-EB. In most patients, TP53 mutations remained detectable in complete remission by deep sequencing (73%). Detection of residual mutant TP53 was not associated with survival. Mutant TP53 AML and MDS-EB do not differ with respect to molecular characteristics and survival. Therefore, mutant TP53 AML/MDS-EB should be considered a distinct molecular disease entity.
•Mutant TP53 AML and MDS-EB do not differ with respect to molecular characteristics and survival.•Mutant TP53 AML/MDS-EB should be considered a single molecular disease entity.
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The applicability of
-internal tandem duplications (
-ITD) for assessing measurable residual disease (MRD) in acute myeloid leukemia (AML) in complete remission (CR) has been hampered by ...patient-specific duplications and potential instability of
-ITD during relapse. Here, we comprehensively investigated the impact of next-generation sequencing (NGS)-based
-ITD MRD detection on treatment outcome in a cohort of patients with newly diagnosed AML in relation to established prognostic factors at diagnosis and other MRD measurements, ie, mutant
and multiparameter flow cytometry.
In 161 patients with de novo
-ITD AML, NGS was performed at diagnosis and in CR after intensive remission induction treatment.
-ITD MRD status was correlated with the cumulative incidence of relapse and overall survival (OS).
NGS-based
-ITD MRD was present in 47 of 161 (29%) patients with AML. Presence of
-ITD MRD was associated with increased risk of relapse (4-year cumulative incidence of relapse, 75%
-ITD MRD
33% no
-ITD MRD;
< .001) and inferior OS (4-year OS, 31%
-ITD MRD
57% no
-ITD MRD;
< .001). In multivariate analysis, detection of
-ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 3.55;
< .001) and OS (hazard ratio 2.51;
= .002). Strikingly,
-ITD MRD exceeds the prognostic value of most generally accepted clinical and molecular prognostic factors, including the
-ITD allelic ratio at diagnosis and MRD assessment by NGS-based mutant
detection or multiparameter flow cytometry.
NGS-based detection of
-ITD MRD in CR identifies patients with AML with profound risk of relapse and death that outcompetes the significance of most established prognostic factors at diagnosis and during therapy, and furnishes support for
-ITD as a clinically relevant biomarker for dynamic disease risk assessment in AML.
Aboriginal Australians are disproportionately affected by dementia, with incidence in remote populations approximately double that of non-Indigenous populations. This study aimed to identify dementia ...incidence and risk factors in Aboriginal Australians residing in urban areas, which are currently unknown.
A population-based cohort of Aboriginal Australians ≥60 years of age was assessed at baseline and 6-year follow-up. Life-course risk factors (baseline) were examined for incident dementia or mild cognitive impairment (MCI) through logistic regression analyses; adjustments were made for age.
genotyping was available for 86 people.
Data were included from 155 participants 60 to 86 years of age (mean 65.70 years, SD 5.65 years; 59 male). There were 16 incident dementia cases (age-standardized rate 35.97/1,000 person-years, 95% confidence interval CI 18.34-53.60) and 36 combined incident MCI and dementia cases. Older age (odds ratio OR 2.29, 95% CI 1.42-3.70), male sex (OR 4.14, 95% CI 1.60-10.77), unskilled work history (OR 5.09, 95% CI 1.95-13.26), polypharmacy (OR 3.11, 95% CI 1.17-8.28), and past smoking (OR 0.24, 95% CI 0.08-0.75) were associated with incident MCI/dementia in the final model.
ε4 allele frequency was 24%; heterozygous or homozygous ε4 was associated with incident MCI/dementia (bivariate OR 3.96, 95% CI 1.25-12.50).
These findings provide evidence for higher dementia incidence in Aboriginal Australians from urban areas, where the majority of Aboriginal people reside. This study also sheds light on sociodemographic, health, and genetic factors associated with incident MCI/dementia at older ages in this population, which is critical for targeted prevention strategies.
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