Advancing national climate change risk assessment to deliver national adaptation plans

A wide range of climate vulnerability and risk assessments have been implemented using different approaches at different scales, some with a broad multi-sectoral scope and others focused on single risks or sectors. This paper describes the novel approach to vulnerability and risk assessment which was designed and put into practice in the United Kingdom's Second Climate Change Risk Assessment (CCRA2) so as to build upon its earlier assessment (CCRA1). First, we summarize and critique the CCRA1 approach, and second describe the steps taken in the CCRA2 approach in detail, providing examples of how each was applied in practice. Novel elements of the approach include assessment of both present day and future vulnerability, a focus on the urgency of adaptation action, and a structure focused around systems of receptors rather than conventional sectors. Both stakeholders and reviewers generally regarded the approach as successful in providing advice on current risks and future opportunities to the UK from climate change, and the fulfilment of statutory duty. The need for a well-supported and open suite of impact indicators going forward is highlighted

A family harboring an MLKL loss of function variant implicates impaired necroptosis in diabetes

Maturity-onset diabetes of the young, MODY, is an autosomal dominant disease with incomplete penetrance. In a family with multiple generations of diabetes and several early onset diabetic siblings, we found the previously reported P33T PDX1 damaging mutation. Interestingly, this substitution was also present in a healthy sibling. In contrast, a second very rare heterozygous damaging mutation in the necroptosis terminal effector, MLKL, was found exclusively in the diabetic family members. Aberrant cell death by necroptosis is a cause of inflammatory diseases and has been widely implicated in human pathologies, but has not yet been attributed functions in diabetes. Here, we report that the MLKL substitution observed in diabetic patients, G316D, results in diminished phosphorylation by its upstream activator, the RIPK3 kinase, and no capacity to reconstitute necroptosis in two distinct MLKL−/− human cell lines. This MLKL mutation may act as a modifier to the P33T PDX1 mutation, and points to a potential role of impairment of necroptosis in diabetes. Our findings highlight the importance of family studies in unraveling MODY’s incomplete penetrance, and provide further support for the involvement of dysregulated necroptosis in human disease.Other Information Published in: Cell Death & Disease License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41419-021-03636-5</p

ESM_RW2 from Advancing national climate change risk assessment to deliver national adaptation plans

Supplementary Material: Worked example and information about UKCP0

High Yield Production of a Soluble Human Interleukin-3 Variant from <i>E. coli</i> with Wild-Type Bioactivity and Improved Radiolabeling Properties

<div><p>Human interleukin-3 (hIL-3) is a polypeptide growth factor that regulates the proliferation, differentiation, survival and function of hematopoietic progenitors and many mature blood cell lineages. Although recombinant hIL-3 is a widely used laboratory reagent in hematology, standard methods for its preparation, including those employed by commercial suppliers, remain arduous owing to a reliance on refolding insoluble protein expressed in <i>E. coli</i>. In addition, wild-type hIL-3 is a poor substrate for radio-iodination, which has been a long-standing hindrance to its use in receptor binding assays. To overcome these problems, we developed a method for expression of hIL-3 in <i>E. coli</i> as a soluble protein, with typical yields of >3mg of purified hIL-3 per litre of shaking microbial culture. Additionally, we introduced a non-native tyrosine residue into our hIL-3 analog, which allowed radio-iodination to high specific activities for receptor binding studies whilst not compromising bioactivity. The method presented herein provides a cost-effective and convenient route to milligram quantities of a hIL-3 analog with wild-type bioactivity that, unlike wild-type hIL‑3, can be efficiently radio-iodinated for receptor binding studies.</p> </div

Purification of the hIL-3(13-125; W13Y) analog.

<div><p>A) Workflow diagram illustrating the purification protocol for hIL-3(13-125; W13Y).</p> <p>B) Elution profile of the TEV protease digested, NusA and hIL-3 analog mixture, following size exclusion chromatography (SEC) using a Superdex 200 column (26 mm x 600 mm) operated at 2 ml/min at 4°C with 50 mM sodium phosphate pH 7.0, 150 mM NaCl as running buffer. Free NusA eluted at ~186 mL and the hIL-3 analog eluted at 254 mL. The first peak at 116 mL contains aggregates while we suspect the last peak at 328 mL contains DTT from the digest. Molecular weight (kDa) marker elution positions are marked as dots above the elution profile.</p> <p>C) Analysis of hIL-3 analog purification by 15% acrylamide reducing SDS-PAGE with Coomassie Blue staining. NusA: hIL-3(13-125; W13Y) fusion protein was isolated by Ni-chromatography (Lane 1) prior to cleavage by TEV protease (Lane 2) to yield free NusA (55kDa) and the hIL-3 analog (13.4kDa). Fractions containing the hIL-3 analog that eluted around 254ml during SEC are shown (Lanes 4-9), illustrating that the hIL-3 analog was purified free from NusA (Lane 3).</p> <p>D) The SEC purified hIL-3 analog was applied to an Aquapore RP300 reversed-phase column (4.6 mm x100 mm) and bound proteins eluted using a 0-100% gradient of acetonitrile in 0.1% trifluoroacetic acid. The hIL-3 analog eluted at 34 min (~50% acetonitrile).</p> <p>E) Purified hIL-3(13-125; W13Y) was subjected to tryptic digestion and tandem mass spectrometry. The sequences of peptides not identified in this analysis are shown as lowercase italics. Asterisked methionine residues were oxidized. Sequence arising from the NusA-His₆ fusion overhang after TEV protease cleavage is underlined, while the residues are numbered according to the mature, full-length IL-3 reference sequence.</p></div

Alignment of amino acid sequences of human and mouse IL-3.

<p>The amino acid sequences of full length wild-type hIL-3, hIL-3(13-125; W13Y), the hIL-3 analog SC-65369 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074376#B41" target="_blank">41</a>], and wild-type full length mouse IL-3 were manually aligned owing to low homology between mouse and human IL-3 (29% identity). Numbers above the sequence refer to the mature form of full length hIL-3 with dots above every 10^th residue. The sequences shown in gray for full length hIL-3 and full length mouse IL-3 are signal peptides that are cleaved during secretion. The key substitution, W13Y in hIL-3(13-125; W13Y), is shown in bold text and highlighted.</p

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N‑Terminus of SOCS5

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5_175–244), consisting of an α-helix encompassing residues 224–233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis

SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target.

<p>SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the <i>K</i>_D values for the respective phosphopeptides. Binding analysis of (<b>A</b>) JAK, Shc-1, or wild-type and (<b>B</b>) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective <i>K</i>_D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. (<b>C</b>) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. (<b>D</b>) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).</p

Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

<div><p>Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.</p></div

An N-terminal fragment corresponding to residues 175–244 of SOCS5 can directly bind JAK1.

<p>(A) SPR analysis of SOCS5^175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5^175–244 protein. Upper panels represent sensorgrams showing the kinetics of binding. Lower panels show steady-state analysis. (B) 293T cells were transfected with the Stat6 reporter and increasing amounts of cDNA expressing Flag-tagged SOCS5 (3.13–100 ng) or SOCS5 lacking the conserved N-terminal fragment (9.5–300 ng; Δ175–244) and stimulated overnight with 10 ng/mL rhIL-4. Cells were lysed and induced luciferase activity measured and normalised according to Renilla activity. Data are expressed as arbitrary units and represent the mean of triplicates ± SD. Cell lysates were analyzed by Western blotting for Flag-tagged proteins (SOCS5 upper; Δ175–244 lower panel); images were generated from the same gel and exposure. (C) Recombinant SOCS5 JIR or SOCS3 was incubated with 20 nM JAK1 and GST-JAK2 activation peptide (substrate; GST-J) for 15 min in the presence of 2.5 mM Mg/³²P-γ-ATP at 37°C. Incorporation of ³²P was visualised by autoradiography (top panel) and protein input by SDS-PAGE and Coomassie staining (lower panel).</p