Trans-activating mutations of the pseudokinase ERBB3.

Genetic changes in the ERBB family of receptor tyrosine kinases serve as oncogenic driver events and predictive biomarkers for ERBB inhibitor drugs. ERBB3 is a pseudokinase member of the family that, although lacking a fully active kinase domain, is well known for its potent signaling activity as a heterodimeric complex with ERBB2. Previous studies have identified few transforming ERBB3 mutations while the great majority of the hundreds of different somatic ERBB3 variants observed in different cancer types remain of unknown significance. Here, we describe an unbiased functional genetics screen of the transforming potential of thousands of ERBB3 mutations in parallel. The screen based on a previously described iSCREAM (in vitro screen of activating mutations) platform, and addressing ERBB3 pseudokinase signaling in a context of ERBB3/ERBB2 heterodimers, identified 18 hit mutations. Validation experiments in Ba/F3, NIH 3T3, and MCF10A cell backgrounds demonstrated the presence of both previously known and unknown transforming ERBB3 missense mutations functioning either as single variants or in cis as a pairwise combination. Drug sensitivity assays with trastuzumab, pertuzumab and neratinib indicated actionability of the transforming ERBB3 variants.

Using emerging science to inform risk characterizations for wildlife within current regulatory frameworks.

Many jurisdictions have regulatory frameworks that seek to reduce the effects of environmental exposures of anthropogenic chemicals on terrestrial wildlife (i.e., mammals, birds, reptiles, and amphibians). The frameworks apply for new and existing chemicals, including pesticides (prospective assessments), and to environmental contamination from releases (retrospective risk assessments). Relatively recently, there have been many scientific advances that could improve risk estimates for wildlife. Here, we briefly describe current regulations from North America (United States and Canada) and from Europe that include risk assessments for wildlife to ascertain whether they are conducive to the use of emerging science and new methods. We also provide examples where new and emerging science may be used to improve wildlife risk characterization and identify areas in need of future research. Integr Environ Assess Manag 2024;20:765-779. © 2024 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management © 2024 Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Integrating emerging science to improve estimates of risk to wildlife from chemical exposure: What are the challenges?

Many jurisdictions require ecological risk assessments for terrestrial wildlife (i.e., terrestrial vertebrates) to assess potential adverse effects from exposure to anthropogenic chemicals. This occurs, for example, at contaminated sites and when new pesticides are proposed, and it occurs for chemicals that are in production and/or proposed for wide-scale use. However, guidance to evaluate such risks has not changed markedly in decades, despite the availability of new scientific tools to do so. In 2019, the Wildlife Toxicology World Interest Group of the Society of Environmental Toxicology and Chemistry (SETAC) initiated a virtual workshop that included a special session coincident with the annual SETAC North America meeting and which focused on the prospect of improving risk assessments for wildlife and improving their use in implementing chemical regulations. Work groups continued the work and investigated the utility of integrating emerging science and novel methods for improving problem formulation (WG1), exposure (WG2), toxicology (WG3), and risk characterization (WG4). Here we provide a summary of that workshop and the follow-up work, the regulations that drive risk assessment, and the key focus areas identified to advance the ability to predict risks of chemicals to wildlife. Integr Environ Assess Manag 2024;20:645-657. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The active site of the SGNH hydrolase-like fold proteins: Nucleophile-oxyanion (Nuc-Oxy) and Acid-Base zones.

SGNH hydrolase-like fold proteins are serine proteases with the default Asp-His-Ser catalytic triad. Here, we show that these proteins share two unique conserved structural organizations around the active site: (1) the Nuc-Oxy Zone around the catalytic nucleophile and the oxyanion hole, and (2) the Acid-Base Zone around the catalytic acid and base. The Nuc-Oxy Zone consists of 14 amino acids cross-linked with eight conserved intra- and inter-block hydrogen bonds. The Acid-Base Zone is constructed from a single fragment of the polypeptide chain, which incorporates both the catalytic acid and base, and whose N- and C-terminal residues are linked together by a conserved hydrogen bond. The Nuc-Oxy and Acid-Base Zones are connected by an SHLink, a two-bond conserved interaction from amino acids, adjacent to the catalytic nucleophile and base.

Toxicological effects assessment for wildlife in the 21st century: Review of current methods and recommendations for a path forward.

Model species (e.g., granivorous gamebirds, waterfowl, passerines, domesticated rodents) have been used for decades in guideline laboratory tests to generate survival, growth, and reproductive data for prospective ecological risk assessments (ERAs) for birds and mammals, while officially adopted risk assessment schemes for amphibians and reptiles do not exist. There are recognized shortcomings of current in vivo methods as well as uncertainty around the extent to which species with different life histories (e.g., terrestrial amphibians, reptiles, bats) than these commonly used models are protected by existing ERA frameworks. Approaches other than validating additional animal models for testing are being developed, but the incorporation of such new approach methodologies (NAMs) into risk assessment frameworks will require robust validations against in vivo responses. This takes time, and the ability to extrapolate findings from nonanimal studies to organism- and population-level effects in terrestrial wildlife remains weak. Failure to adequately anticipate and predict hazards could have economic and potentially even legal consequences for regulators and product registrants. In order to be able to use fewer animals or replace them altogether in the long term, vertebrate use and whole organism data will be needed to provide data for NAM validation in the short term. Therefore, it is worth investing resources for potential updates to existing standard test guidelines used in the laboratory as well as addressing the need for clear guidance on the conduct of field studies. Herein, we review the potential for improving standard in vivo test methods and for advancing the use of field studies in wildlife risk assessment, as these tools will be needed in the foreseeable future. Integr Environ Assess Manag 2024;20:699-724. © 2023 His Majesty the King in Right of Canada and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Comprehensive characterization of the embryonic factor LEUTX.

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.

Integrating "One Health" Concepts in the Design of Sustainable Systems for Environmental Use.

Ensuring for the national defense requires the use of substances such as energetics, propellants, pyrotechnics, and other materials in environmental applications. Systems that use these materials do so in testing and training environments and must be used in an environmentally sustained manner to ensure success during actual kinetic defensive operations. Environmental and occupational health assessments require a weighted evaluation of toxicity, bioaccumulation, persistence, and environmental fate and transport considerations for each substance in the formulation to include potential combustion products. Data that support these criteria need to be collected in a phased and matrixed approach and considered iteratively as technology advances. Further, these criteria are often considered as disparate and separate; hence, comparing favorable aspects of one may or may not offset detrimental data from another. Here, we describe an approach to the phased collection of environmental, safety, and occupational health (ESOH) information for new systems and substances and provide recommendations for evaluating such data streams in making decisions for use and for evaluating alternatives.

An Educational Intervention to Enhance Palliative Care Training at HBCUs.

CONTEXT: Primary palliative care training is important for clinicians at Historically Black Colleges and Universities (HBCUs) given the decreased access to specialty palliative care among Black patients and patients' preferences for race concordant care. OBJECTIVES: To describe the impact of a palliative care educational intervention at two HBCUs. METHODS: We administered a palliative care educational intervention in family and internal medicine residency programs at Morehouse School of Medicine and Howard University College of Medicine. Pre- and post-intervention surveys were sent to residents assessing attitudes toward their palliative care education and their perceived competency in specific palliative care domains. The results were analyzed using Chi-squared analysis. RESULTS: A total of 105 of 191 (response rate 55%) residents completed pre-intervention surveys and 101 of 240 (42%) completed post-intervention surveys. Prior to the intervention, 50% of residents rated their overall preparedness in palliative care as a 7 or above (0-10 Likert scale); 78% (P < .01) of respondents reported ≥7/10 after the educational intervention. While post-intervention residents did not feel better prepared to treat symptoms, a higher percentage reported feeling well prepared to deliver bad news (41% post-intervention vs. 23% pre-intervention) and conduct a family meeting (44% post-intervention vs. 27% pre-intervention) (P < .05). Pre-intervention, 14% of residents felt their overall palliative care education was very good or excellent, while post-intervention ratings increased to 30% (P < .01). CONCLUSION: Residents' confidence in their preparedness to provide palliative care, particularly in their communication skills increased after an intervention at two HBCUs.

Canonical structural-binding modes in the calmodulin-target protein complexes.

Intracellular calcium sensor protein calmodulin (CaM) belongs to the large EF-hand protein superfamily. CaM shows a unique and not fully understood ability to bind to multiple targets, allows them to participate in a variety of regulatory processes. The protein has two approximately symmetrical globular domains (the N- and C-lobes). Analysis of the CaM-binding sites of target proteins showed that they have two hydrophobic 'anchor' amino acids separated by 10 to 17 residues. Consequently, several CaM-binding motifs: {1-10}, {1-11}, {1-13}, {1-14}, {1-16}, {1-17}, differing by the distance between the two anchor residues along the amino acid sequence, have been identified. Despite extensive structural information on the role of target-protein amino acid residues in the formation of complexes with CaM, much less is known about the role of amino acids from CaM contributing to these interactions. In this work, a quantitative analysis of the contact surfaces of CaM and target proteins has been carried out for 35 representative three-dimensional structures. It has been shown that, in addition to the two hydrophobic terminal residues of the target fragment, the interaction also involves residues that are 4 residues earlier in the sequence (binding mode {1-5}). It has also been found that the N- and C-lobes of CaM bind the {1-5} motif located at the ends of the target in a structurally identical manner. Methionine residues at positions 51 (corresponding to 124 in the C-lobe), 71 (144), and 72 (145) of the CaM amino acid sequence are key hydrophobic residues for this interaction. They are located at the N- and C-boundaries of the even EF-hand motifs. The hydrophobic core of CaM ('Ф-quatrefoil') consists of 10 amino acids in the N-lobe (and in the C-lobe): Phe16 (Phe89), Phe19 (Phe92), Ile27 (Ile100), Thr29 (Ala102), Leu32 (Leu105), Ile52 (Ile125), Val55 (Ala128), Ile63 (Val136), Phe65 (Tyr138), and Phe68 (Phe141) and do not intersect with the target-binding methionine residues. CaM belongs to the 'dynamic' group of EF-hand proteins, in which calcium and protein ligand binding causes only global conformational changes but does not alter the conservative 'black' and 'grey' clusters described in our earlier works (PLoS One. 2014; 9(10):e109287). The membership of CaM in the 'dynamic' group is determined by the triggering and protective methionine layer: Met51 (Met124), Met71 (Met144) and Met72 (Met145). HIGHLIGHTSInterchain interactions in the unique 35 CaM complex structures were analyzed.Methionine amino acids of the N- and C-lobes of CaM form triggering and protective layers.Interactions of the target terminal residues with these methionine layers are structurally identical.CaM belonging to the 'dynamic' group is determined by the triggering and protective methionine layer.Communicated by Ramaswamy H. Sarma.

Key challenges and developments in wildlife ecological risk assessment: Problem formulation.

Problem formulation (PF) is a critical initial step in planning risk assessments for chemical exposures to wildlife, used either explicitly or implicitly in various jurisdictions to include registration of new pesticides, evaluation of new and existing chemicals released to the environment, and characterization of impact when chemical releases have occurred. Despite improvements in our understanding of the environment, ecology, and biological sciences, few risk assessments have used this information to enhance their value and predictive capabilities. In addition to advances in organism-level mechanisms and methods, there have been substantive developments that focus on population- and systems-level processes. Although most of the advances have been recognized as being state-of-the-science for two decades or more, there is scant evidence that they have been incorporated into wildlife risk assessment or risk assessment in general. In this article, we identify opportunities to consider elevating the relevance of wildlife risk assessments by focusing on elements of the PF stage of risk assessment, especially in the construction of conceptual models and selection of assessment endpoints that target population- and system-level endpoints. Doing so will remain consistent with four established steps of existing guidance: (1) establish clear protection goals early in the process; (2) consider how data collection using new methods will affect decisions, given all possibilities, and develop a decision plan a priori; (3) engage all relevant stakeholders in creating a robust, holistic conceptual model that incorporates plausible stressors that could affect the targets defined in the protection goals; and (4) embrace the need for iteration throughout the PF steps (recognizing that multiple passes may be required before agreeing on a feasible plan for the rest of the risk assessment). Integr Environ Assess Manag 2022;00:1-16. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation.

The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as ß1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.

An Evolutionary Perspective on the Origin, Conservation and Binding Partner Acquisition of Tankyrases.

Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species may exist. In this work, we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identify the single-celled choanoflagellates as earliest origin of tankyrases. We further show that the sequences and structural aspects of TNKSs are well-conserved even between distantly related species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners varies greatly between species, indicating that tankyrases may have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, and that their regulatory functions in cells have likely changed considerably throughout evolution.

DUX4 is a multifunctional factor priming human embryonic genome activation.

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

Examining the Effect of Charged Lipids on Mitochondrial Outer Membrane Dynamics Using Atomistic Simulations.

The outer mitochondrial membrane (OMM) is involved in multiple cellular functions such as apoptosis, inflammation and signaling via its membrane-associated and -embedded proteins. Despite the central role of the OMM in these vital phenomena, the structure and dynamics of the membrane have regularly been investigated in silico using simple two-component models. Accordingly, the aim was to generate the realistic multi-component model of the OMM and inspect its properties using atomistic molecular dynamics (MD) simulations. All major lipid components, phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), were included in the probed OMM models. Because increased levels of anionic PS lipids have potential effects on schizophrenia and, more specifically, on monoamine oxidase B enzyme activity, the effect of varying the PS concentration was explored. The MD simulations indicate that the complex membrane lipid composition (MLC) behavior is notably different from the two-component PC-PE model. The MLC changes caused relatively minor effects on the membrane structural properties such as membrane thickness or area per lipid; however, notable effects could be seen with the dynamical parameters at the water-membrane interface. Increase of PS levels appears to slow down lateral diffusion of all lipids and, in general, the presence of anionic lipids reduced hydration and slowed down the PE headgroup rotation. In addition, sodium ions could neutralize the membrane surface, when PI was the main anionic component; however, a similar effect was not seen for high PS levels. Based on these results, it is advisable for future studies on the OMM and its protein or ligand partners, especially when wanting to replicate the correct properties on the water-membrane interface, to use models that are sufficiently complex, containing anionic lipid types, PI in particular.

The Biomolecules Journal Club: Highlights on Recent Papers-1.

We are glad to share with you our first Journal Club and to highlight some of the most interesting papers published recently [...].

An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations.

Despite the relatively high frequency of somatic ERBB4 mutations in various cancer types, only a few activating ERBB4 mutations have been characterized, primarily due to lack of mutational hotspots in the ERBB4 gene. Here, we utilized our previously published pipeline, an in vitro screen for activating mutations, to perform an unbiased functional screen to identify potential activating ERBB4 mutations from a randomly mutated ERBB4 expression library. Ten potentially activating ERBB4 mutations were identified and subjected to validation by functional and structural analyses. Two of the 10 ERBB4 mutants, E715K and R687K, demonstrated hyperactivity in all tested cell models and promoted cellular growth under two-dimensional and three-dimensional culture conditions. ERBB4 E715K also promoted tumor growth in in vivo Ba/F3 cell mouse allografts. Importantly, all tested ERBB4 mutants were sensitive to the pan-ERBB tyrosine kinase inhibitors afatinib, neratinib, and dacomitinib. Our data indicate that rare ERBB4 mutations are potential candidates for ERBB4-targeted therapy with pan-ERBB inhibitors. Statement of Significance: ERBB4 is a member of the ERBB family of oncogenes that is frequently mutated in different cancer types but the functional impact of its somatic mutations remains unknown. Here, we have analyzed the function of over 8,000 randomly mutated ERBB4 variants in an unbiased functional genetics screen. The data indicate the presence of rare activating ERBB4 mutations in cancer, with potential to be targeted with clinically approved pan-ERBB inhibitors.

Structural and functional significance of the amino acid differences Val35Thr, Ser46Ala, Asn65Ser, and Ala94Ser in 3C-like proteinases from SARS-CoV-2 and SARS-CoV.

Three dimensional structures of (chymo)trypsin-like proteinase (3CLpro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val86Leu, Lys88Arg, Phe134His, and Asn180Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CLpro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val35Thr, Ser46Ala, Asn65Ser, Ala94Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CLpro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CLpro(s) can use for functional regulation an autolytic loop and the unique Asp33-Asn95 region (the Asp33-Asn95 Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CLpro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.

Advances in Assessing Hazard and Risk to Emerging Threats and Emergency Response: Comparing and Contrasting Efforts of 3 Federal Agencies.

Federal statutes authorize several agencies to protect human populations from chemical emergencies and provide guidance to evacuate, clean, and reoccupy affected areas. Each of the authorized federal agencies has developed programs to provide managers, public health officials, and regulators, with a rapid assessment of potential hazards and risks associated with chemical emergencies. Emergency responses vary based on exposure scenarios, routes, temporal considerations, and the substance(s) present. Traditional chemical assessments and derivation of toxicity values are time-intensive, typically requiring large amounts of human epidemiological and experimental animal data. When a rapid assessment of health effects is needed, an integrated computational approach of augmenting extant toxicity data with in vitro (new alternative toxicity testing methods) data can provide a quick, evidence-based solution. In so doing, multiple streams of data can be used, including literature searches, hazard, dose-response, physicochemical, environmental fate, transport property data, in vitro cell bioactivity testing, and toxicogenomics. The field of toxicology is moving, towards increased use of this approach as it transforms from observational to predictive science. The challenge is to objectively and transparently derive toxicity values using this approach to protect human health and the environment. Presented here are examples and efforts toward rapid risk assessment that demonstrate unified, parallel, and complementary work to provide timely protection in times of chemical emergency.

Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.