Evaluation of MRN complex and ATM protein-protein interactions using cleavable DSSO crosslinking and mass spectrometry

Ataxia telangiectasia (A–T) is an inherited autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The canonical role of ATM is to induce cell checkpoint arrest following DNA damage. When cells experience DNA double-strand breaks (DSBs), the Mre11-Rad50-Nbs1 (MRN) complex senses the damage, activating ATM, which mobilizes a protective signaling cascade, activating the cell cycle checkpoint, arresting cell growth. If the extent of damage is excessive, the apoptotic pathway is activated; otherwise DNA repair is initiated using either homologous recombination (HR) or through non-homologous end joining (NHEJ). ATM also plays a key role in redox homeostasis. Loss of this cellular function results in the misfolding and aggregation of proteins. Misfolded proteins are degraded by three pathways: the ubiquitin proteasome system (UPS), macroautophagy and chaperone-mediated autophagy (CMA). Aggregated proteins that are resistant to these protein clearance mechanisms can form inclusion bodies, which can lead to neurodegeneration. The primary approach used for investigating protein interactions in this research was with the DSSO cleavable crosslinking technique: first to evaluate the MRN protein complex structure and protein-protein interactions; then to elucidate ATM binding partners in cells undergoing oxidative stress. The results of the MRN complex in vitro crosslinking experiment were that 53 unique crosslinks were detected. These were subsequently evaluated by calculating molecular distances between identified crosslinked residues using known crystal structures of homologous proteins. Conversely, the results of the ATM in vivo crosslinking experiment were that, while informative crosslinks were not detected between ATM and other proteins, certain pathways were over-, and under-represented in the Co-IP of cells expressing the ATM constructs. Specifically, the parkin-ubiquitin proteasomal system pathway was over-represented, while the proteasome degradation pathway was under-represented. Additionally, the small heat shock protein 27 (Hsp27) was found to be enriched, a chaperone known for its protective role in protein aggregation diseases.Cellular and Molecular Biolog

Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses

Mice engrafted with components of a human immune system have become widely-used models for studying aspects of human immunity and disease. However, a defined methodology to objectively measure and compare the quality of the human immune response in different models is lacking. Here, by taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we provide an in-depth comparison of immune responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells promotes transcriptomic responses akin to those of human vaccinees. These enhanced transcriptomic profiles correlate with the development of an antigen-specific cellular and humoral response to YFV-17D. Altogether, our approach provides a robust scoring of the quality of the human immune response in humanized mice and highlights a rational path towards developing better pre-clinical models for studying the human immune response and disease.National Institute of General Medical Sciences (U.S.) (Award T32GM007753)Searle Scholars ProgramArnold and Mabel Beckman Foundation (Young Investigator Program)National Institutes of Health (U.S.) (1DP2OD020839)National Institutes of Health (U.S.) (5U24AI118672)National Institutes of Health (U.S.) (1U54CA217377)National Institutes of Health (U.S.) (1R33CA202820)National Institutes of Health (U.S.) (2U19AI089992)National Institutes of Health (U.S.) (21R01HL134539)National Institutes of Health (U.S.) (2RM1HG006193)National Institutes of Health (U.S.) (2R01HL095791)National Institutes of Health (U.S.) (P01AI039671)Bill & Melinda Gates Foundation (OPP1139972

ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

: The transcription factor ETV7 is an oncoprotein that is up-regulated in all breast cancer (BC) types. We have recently demonstrated that ETV7 promoted breast cancer progression by increasing cancer cell proliferation and stemness and was also involved in the development of chemo- and radio-resistance. However, the roles of ETV7 in breast cancer inflammation have yet to be studied. Gene ontology analysis previously performed on BC cells stably over-expressing ETV7 demonstrated that ETV7 was involved in the suppression of innate immune and inflammatory responses. To better decipher the involvement of ETV7 in these signaling pathways, in this study, we identified TNFRSF1A, encoding for the main receptor of TNF-α, TNFR1, as one of the genes down-regulated by ETV7. We demonstrated that ETV7 directly binds to the intron I of this gene, and we showed that the ETV7-mediated down-regulation of TNFRSF1A reduced the activation of NF-κB signaling. Furthermore, in this study, we unveiled a potential crosstalk between ETV7 and STAT3, another master regulator of inflammation. While it is known that STAT3 directly up-regulates the expression of TNFRSF1A, here we demonstrated that ETV7 reduces the ability of STAT3 to bind to the TNFRSF1A gene via a competitive mechanism, recruiting repressive chromatin remodelers, which results in the repression of its transcription. The inverse correlation between ETV7 and TNFRSF1A was confirmed also in different cohorts of BC patients. These results suggest that ETV7 can reduce the inflammatory responses in breast cancer through the down-regulation of TNFRSF1A

TMX2 Is a Crucial Regulator of Cellular Redox State, and Its Dysfunction Causes Severe Brain Developmental Abnormalities.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain