A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.

Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase

Regional Invasive Species & Climate Change Management Challenge: Gardening with climate-smart native plants in the Northeast

An estimated 80% of ornamental plants for sale are non-native. This means that the average yard does a poor job of supporting native flora and fauna. By shifting our plantings towards natives, we can dramatically increase the diversity of bees, butterflies, birds and other animals. In contrast, non-native plants do not support local food webs and can become invasive. Native plants increase biodiversity and reduce risks associated with invasive species, which supports resilient ecosystems in the face of climate change

SARS-CoV-2 omicron (B.1.1.529)-related COVID-19 sequelae in vaccinated and unvaccinated patients with cancer: results from the OnCovid registry

Background COVID-19 sequelae can affect about 15% of patients with cancer who survive the acute phase of SARS-CoV-2 infection and can substantially impair their survival and continuity of oncological care. We aimed to investigate whether previous immunisation affects long-term sequelae in the context of evolving variants of concern of SARS-CoV-2. Methods OnCovid is an active registry that includes patients aged 18 years or older from 37 institutions across Belgium, France, Germany, Italy, Spain, and the UK with a laboratory-confirmed diagnosis of COVID-19 and a history of solid or haematological malignancy, either active or in remission, followed up from COVID-19 diagnosis until death. We evaluated the prevalence of COVID-19 sequelae in patients who survived COVID-19 and underwent a formal clinical reassessment, categorising infection according to the date of diagnosis as the omicron (B.1.1.529) phase from Dec 15, 2021, to Jan 31, 2022; the alpha (B.1.1.7)-delta (B.1.617.2) phase from Dec 1, 2020, to Dec 14, 2021; and the pre-vaccination phase from Feb 27 to Nov 30, 2020. The prevalence of overall COVID-19 sequelae was compared according to SARS-CoV-2 immunisation status and in relation to post-COVID-19 survival and resumption of systemic anticancer therapy. This study is registered with ClinicalTrials.gov, NCT04393974. Findings At the follow-up update on June 20, 2022, 1909 eligible patients, evaluated after a median of 39 days (IQR 24-68) from COVID-19 diagnosis, were included (964 [ 50 center dot 7%] of 1902 patients with sex data were female and 938 [49 center dot 3%] were male). Overall, 317 (16 center dot 6%; 95% CI 14 center dot 8-18 center dot 5) of 1909 patients had at least one sequela from COVID-19 at the first oncological reassessment. The prevalence of COVID-19 sequelae was highest in the prevaccination phase (191 [19 center dot 1%; 95% CI 16 center dot 4-22 center dot 0] of 1000 patients). The prevalence was similar in the alpha-delta phase (110 [16 center dot 8%; 13 center dot 8- 20 center dot 3] of 653 patients, p=0 center dot 24), but significantly lower in the omicron phase (16 [6 center dot 2%; 3 center dot 5-10 center dot 2] of 256 patients, p<0 center dot 0001). In the alpha- delta phase, 84 (18 center dot 3%; 95% CI 14 center dot 6-22 center dot 7) of 458 unvaccinated patients and three (9 center dot 4%; 1 center dot 9- 27 center dot 3) of 32 unvaccinated patients in the omicron phase had sequelae. Patients who received a booster and those who received two vaccine doses had a significantly lower prevalence of overall COVID-19 sequelae than unvaccinated or partially vaccinated patients (ten [7 center dot 4%; 95% CI 3 center dot 5-13 center dot 5] of 136 boosted patients, 18 [9 center dot 8%; 5 center dot 8-15 center dot 5] of 183 patients who had two vaccine doses vs 277 [ 18 center dot 5%; 16 center dot 5-20 center dot 9] of 1489 unvaccinated patients, p=0 center dot 0001), respiratory sequelae (six [4 center dot 4%; 1 center dot 6-9 center dot 6], 11 [6 center dot 0%; 3 center dot 0-10 center dot 7] vs 148 [9 center dot 9%; 8 center dot 4- 11 center dot 6], p= 0 center dot 030), and prolonged fatigue (three [2 center dot 2%; 0 center dot 1-6 center dot 4], ten [5 center dot 4%; 2 center dot 6-10 center dot 0] vs 115 [7 center dot 7%; 6 center dot 3-9 center dot 3], p=0 center dot 037)

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

NF-[kappa]B RelB/p52 heterodimer : biogenesis and DNA recognition

NF-[kappa]B is a family of dimeric transcription factors made up of five family members, p50 (NF-[kappa]B1), RelA (p65), p52 (NF-[kappa]B2), c-Rel and RelB, that regulate the expression of a large number of genes involved in innate and adaptive immunity. The NF-[kappa]B dimers regulate gene expression by binding to a class of decameric DNA duplexes known as [kappa]B sites. RelB is the most poorly understood and unique member of the NF- [kappa]B family. Unlike other members, RelB does not form a stable detectable homodimer in vivo. In uninduced cells, RelB preferentially associates with p100 and to a lesser extent with p50. Gene knockout studies demonstrated that RelB and p52 are involved in common biological functions such as secondary lymphoid organ development and B-cell maturation. It was subsequently found that these two proteins act together as a heterodimeric transcription factor that activates a set of genes in the developmental programs. The promoters of several of these genes have shown to contain [kappa]B sites that somewhat deviate from the classical [kappa]B sites. There are two important questions that remain to be answered. These questions are 1) how is the RelB/p52 heterodimer generated and 2) how does this heterodimer bind to [kappa]B sites? The purpose of this dissertation was to provide answers to these questions. The RelB/p52 heterodimer is the product of NF- [kappa]B signaling pathways, referred to as the non- canonical pathways, which involve processing of the precursor protein p100 into p52. However, whether the RelB /p52 heterodimer is generated from the p100/RelB complex observed in uninduced cells was unclear. Results shown in this dissertation demonstrate that NF-[kappa]B RelB is an unstable protein that is stabilized by forming complexes with both p100 and p52. The p100/RelB complex involves all functional domains of each protein and is not processing competent. A large body of experimental results shown here clearly suggests that p52 is primarily generated from newly synthesized p100 and not from the pre-existing p100/ RelB complex. Processing of the newly synthesized p100 is facilitated when bound to a newly synthesized NF-[kappa]B subunit, including RelB. However, the binding of newly synthesized p100 with RelB can also lead to the formation of an inactive p100/RelB complex. Therefore a dynamic binding mode between p100 and RelB, where one is processing competent and the other is not, is proposed. To understand the [kappa]B DNA recognition by the RelB/p52 heterodimer, the complex between the heterodimer and [kappa]B DNA has been crystallized and the 3.05 Å crystal structure is reported in this dissertation. The structure has revealed a new mode of DNA interaction by an NF- [kappa]B dimer that has never been observed previously. In addition to the conserved DNA contacts made by all NF- [kappa]B family members, a conserved arginine of RelB contacts the outermost G:C base pair of the RelB DNA half site. At the same time RelB loses one contact with the innermost A:T base pair of the half site normally mediated by a conserved tyrosine residue. Binding affinity measurements demonstrate that the RelB/p52 heterodimer binds to the [kappa]B DNA tested, including the newly identified ones indicated above, with similar affinities. We propose that the RelB/p52 heterodimer binds to different [kappa]B sites with high affinity by altering the DNA contact. The RelB subunit allows for different binding modes by binding to some [kappa]B sequences using the tyrosine contacts and to other [kappa]B sequences by utilizing the arginine contact. Specific DNA sequences at the center and flanking region dictate the RelB binding mode

NF-κB p52:RelB heterodimer recognizes two classes of κB sites with two distinct modes

The X-ray structure of the nuclear factor-κB (NF-κB) p52:RelB:κB DNA complex reveals a new recognition feature not previously seen in other NF-κB:κB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of κB sites. We found that the Arg 125-sensitive κB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein-induced kink observed in this complex, which used an AT-rich κB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non-AT-rich κB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of κB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in κB sites when compared with other NF-κB dimers

Oligomerization of hIKK2 dimers.

<p>(A) Ribbon diagram of the interaction between two neighboring hIKK2 dimers in the crystal. Their asymmetric association gives rise to two unique intersubunit interfaces. (B) Close-up view of residues that interact between the KDs at the V-shaped interface. (C) Additional residues that mediate V-shaped interface interactions between the ULD an SDD. (D) Close-up view of interacting residues within the anti-parallel interface. (E) In vitro kinase assay reveals that catalytic activity of hIKK2 with mutations that disrupt the V-shaped interface (lanes 3–5) is drastically reduced compared to wild-type protein (WT-lane 2). (F) In vitro kinase assays with the same WT mutant proteins in which activation loop serines are mutated to glutamate. (G) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the V-shaped interface mutants correlates with activation loop phosphorylation status. (H) In vitro kinase assays reveal the modest effects on hIKK2 catalytic activity of mutation at the antiparallel interface.</p

Interaction between KDs of oligomeric hIKK2.

<p>(A) Within the crystal, neighboring tetrameric assemblies interact symmetrically such that they contact one another through their V-shaped interfaces and two KDs are positioned within close proximity to one another (dashed box). (B) The close packed KDs are positioned so that their activation loops (dashed box) rest directly over the active site of a neighbor. Orange spheres mark the Cα positions on V229 and H232. (C) Close-up view of the kinase activation loops (yellow and blue) with glutamic acid residues 177 and 181 mimicking activation loop serines and the catalytic base D145 labeled. (D) In vitro kinase assay on immunoprecipitated hIKK2 with mutations at key residues that mediate KD–KD interactions in the crystal (lanes 3,4) reveals their involvement in catalytic activity. (E) Mutation of activation loop serines 177 and 181 to glutamates restores activity of immunoprecipitated IKK2 in vitro. (F) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the KD–KD interface mutants correlates with decreased activation loop phosphorylation.</p

In vitro reconstitution of hIKK2 <i>trans</i> auto-phosphorylation.

<p>A catalytically inactive (D145N) and C-terminally truncated IKK2 (lanes 1–6) and mixtures of that enzyme with a catalytically active full-length version (lanes 4–6) were incubated with Mg-ATP for the time periods indicated and then probed via Western blot with anti-phosphoSer181 antibody (above) or by SDS PAGE (below).</p