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The adaptor protein Swiprosin-1/Efhd2 that we identified recently in B cell lipid rafts exhibits its highest expression in immature B cells. Swiprosin-1/Efhd2 consists of a proline rich domain, two EF-hands and a C-terminal coiledcoil domain. Knock-down of Swiprosin-1/Efhd2 in an immature B cell line with a shRNA directed against the 3'-UTR (WEHI231shSw1) impaired spontaneous and BCR-elicited apoptosis. Due to co-clustering of a Swiprosin-1-EGFP fusion protein with the B-cell receptor we expected Swiprosin-1 to be a player in proximal BCR signaling. Indeed, downregulation of Swiprosin-1 attenuated total tyrosine phosphorylation and diminished BCR induced intracellular calcium flux. In contrast, WEHI231 cells ectopically expressing Swiprosin-1 showed increased, but shortened total tyrosine phosphorylation at early time points, as well as increased BCR induced calcium flux. Concomitantly, transient re-expression of Swiprosin-1 in WEHI231shSW1 cells restored BCR-induced calcium flux. These data suggested interactions of Swiprosin-1 with central elements of the BCR signaling cascade. In fact, GST-pulldown experiments showed that Swiprosin-1 can interact with tyrosine-phosphorylated proteins of ~145 and 70 kDa. With this assay we identified the protein tyrosine kinase Syk in its phosphorylated form as interaction partner of Swiprosin-1. The interaction of pSyk with Swiprosin-1 is mediated by the C-terminal part of Swiprosin-1 because a deletion mutant of Swiprosin-1 lacking the EF-hands and the coiled-coil domain of Swiprosin-1 did not show any interaction with pSyk. We next tested the kinase activity of Syk as a function of Swiprosin-1 levels. Interestingly, immune complex kinase assays showed that BCR-induced Syk activity was reduced in WEHI231shSw1 cells Hence, Swiprosin-1 could regulate proximal BCR signaling through maintenance of Syk activity. [ABSTRACT FROM AUTHOR]

Carma-1 is required for B cell receptor-/CD40- and T cell receptor-/CD28-induced B- and T-cell activation via JNK and NF-βB. In B cells, Carma-1 becomes phosphorylated by PKCβ, leading to its oligomerization. Subsequent Bcl10 binding induces IKKβ-activation and, thereby, canonical NF-κB signalling. Despite these findings it is still unknown how exactly Carma-1 is connected to the plasma membrane and to the IKK-complex. Therefore, we purified Carma-1 complexes from mouse CH12 B cells using anti-Carma-1 affinity columns. Mass spectrometric analyses of the column eluates demonstrated the presence of Carma-1 as well as three previously uncharacterized adaptor proteins in B cells, one of which was the Trk-fused gene (Tfg), an adaptor protein containing PB1 and coiledcoil domains. Whereas Tfg was originally identified as fusion partner of oncogenic Trk tyrosine kinase mutants, the normal cellular homologue of Tfg has so far not been described in B cells. However, Tfg has been shown in other systems to interact with IKKγ and to enhance TNF-induced NF-κB activation. Tfg and Carma-1 co-localized at the plasma membrane and perinuclear structures in B cells. We further corroborated the interactions of Tfg, IKKγ and Carma-1 by Blue Native gel electrophoresis, where Carma-1 and Tfg formed a 0.7-1 MDa complex. Ectopic expression of Tfg increased the molecular mass of IKKγ complexes, fused IKKγ, Bcl10 and Carma-1 complexes to a ~2 MDa complex, and increased basal and CD40-induced canonical activity of NF-κB and IKKβ. In contrast, shRNA-mediated silencing of Tfg decreased CD40-induced IKKβ activity. Very interestingly, in primary B cells, highest expression of Tfg was detected in marginal zone and B1 B cells, and Carma-1 and Tfg formed complexes in these B cells. Since Carma-1 is required for marginal zone B cell and B1 B cell development, we suggest that a functional interaction between Carma-1 and Tfg contributes to development and maintenance of these cells by means of canonical NF-κB signals. [ABSTRACT FROM AUTHOR]

Background: Hypohidrotic ectodermal dysplasia (HED) is a group of genodermatoses in which deficient ectodysplasin A signalling leads to maldevelopment of skin appendages, various eccrine glands, and teeth. Individuals with HED often have disrupted epithelial barriers and, therefore, were suspected to be more susceptible to coronavirus infection., Methods: 56 households with at least one member who had coronavirus disease of 2019 (COVID-19) were enrolled in a longitudinal study to compare the course of illness, immune responses, and long-term consequences of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection in HED patients (n = 15, age 9-52 years) and control subjects of the same age group (n = 149)., Results: In 14 HED patients, mild or moderate typical COVID-19 symptoms were observed that lasted for 4-45 days. Fever during the first days sometimes required external cooling measures. The course of COVID-19 was similar to that in control subjects if patients developed antibodies blocking the SARS-CoV-2 spike protein. Five out of six HED patients with completely abrogated ectodysplasin A signalling (83%) suffered from chronic, in two cases very severe fatigue following COVID-19, while only 25% of HED patients with residual activity of this pathway and 21% of control subjects recovering from COVID-19 experienced postinfectious fatigue. Hair loss after COVID-19 was also more frequent among HED patients (64%) than in the control group (13%)., Conclusions: HED appears to be associated with an increased risk of long-term consequences of a SARS-CoV-2 infection. Preventive vaccination against COVID-19 should be recommended for individuals affected by this rare genetic disorder., (© 2021. The Author(s).)

Background: The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures., Methods: Gene fitness was determined by bioinformatics analysis from available datasets from genetic screenings. HSPD1 expression was evaluated by immunohistochemistry from formalin-fixed paraffin-embedded tissues from NSCLC patients. Real-time proliferation assays with and without cytotoxicity reagents, colony formation assays and cell cycle analyses were used to monitor growth and drug sensitivity of different NSCLC cells in vitro. In vivo growth was monitored with subcutaneous injections in immune-deficient mice. Cell metabolic activity was analyzed through extracellular metabolic flux analysis. Specific knockouts were introduced by CRISPR/Cas9., Results: We show heat shock protein family D member 1 (HSPD1 or HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients' prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on oxidative phosphorylation and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B., Conclusions: These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NSCLC., (© 2021. The Author(s).)

Changes in the intracellular calcium concentration govern cytoskeletal rearrangement, mitosis, apoptosis, transcriptional regulation or synaptic transmission, thereby, regulating cellular effector and organ functions. Calcium binding proteins respond to changes in the intracellular calcium concentration with structural changes, triggering enzymatic activation and association with downstream proteins. One type of calcium binding proteins are EF-hand super family proteins. Here, we describe two recently discovered homologous EF-hand containing adaptor proteins, Swiprosin-1/EF-hand domain containing 2 (EFhd2) and Swiprosin-2/EF-hand domain containing 1 (EFhd1), which are related to allograft inflammatory factor-1 (AIF-1). For reasons of simplicity and concision we propose to name Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 from now on EFhd2 and EFhd1, according to their respective gene symbols. AIF-1 and Swiprosin-1/EFhd2 are already present in Bilateria, for instance in Drosophila melanogaster and Caenhorhabditis elegans. Swiprosin-2/EFhd1 arose later from gene duplication in the tetrapodal lineage. Secondary structure prediction of AIF-1 reveals disordered regions and one functional EF-hand. Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 exhibit a disordered region at the N-terminus, followed by two EF-hands and a coiled-coil domain. Whereas both proteins are similar in their predicted overall structure they differ in a non-homologous stretch of 60 amino acids just in front of the EF-hands. AIF-1 controls calcium-dependent cytoskeletal rearrangement in innate immune cells by means of its functional EF-hand. We propose that Swiprosin-1/EFhd2 as well is a cytoskeleton associated adaptor protein involved in immune and brain cell function. Pro-inflammatory conditions are likely to modulate expression and function of Swiprosin-1/EFhd2. Swiprosin-2/EFhd1, on the other hand, modulates apoptosis and differentiation of neuronal and muscle precursor cells, probably through an association with mitochondria. We suggest furthermore that Swiprosin-2/EFhd1 is part of a cellular response to oxidative stress, which could explain its pro-survival activity in neuronal, muscle and perhaps some malignant tissues.