Transmembrane Complexes of DAP12 Crystallized in Lipid Membranes Provide Insights into Control of Oligomerization in Immunoreceptor Assembly

The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces

Tetraspanin CD151 plays a key role in skin squamous cell carcinoma

Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size, and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared to other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3, a regulator of cell proliferation and apoptosis. CD151 also supports PKCα-α6β4 integrin association and PKC-dependent β4 S1424 phosphorylation, while regulating α6β4 distribution. CD151-PKCα effects on integrin β4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including DMBA (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting EGFR, PKC, Jak2/Tyk2, and STAT3. Hence, CD151 ‘co-targeting’ may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151-laminin-binding integrin complexes

Fibroblastic reticular cells provide a supportive niche for lymph node–resident macrophages

The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis, but key factors controlling this niche are undefined. Here, we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Genetic ablation of FRCs caused rapid loss of macrophages and monocytes from LNs across two in vivo models. Macrophages co‐localized with FRCs in human LNs, and murine single‐cell RNA‐sequencing revealed that FRC subsets broadly expressed master macrophage regulator CSF1. Functional assays containing purified FRCs and monocytes showed that CSF1R signaling was sufficient to support macrophage development. These effects were conserved between mouse and human systems. These data indicate an important role for FRCs in maintaining the LN parenchymal macrophage niche

Functional characterisation of the N-terminal domain of polyomavirus large T antigen

Although scientists have extensively researched the relationship between viral oncoproteins and cellular tumour suppressor proteins in recent years, the molecular interactions between these proteins is still poorly understood. It is the goal of this thesis to establish the key elements of specific interactions, in particular to characterise the interaction between the N-terminal part of the viral murine polyoma oncoprotein large T antigen (PyLTNT), and the cellular human regulator protein retinoblastoma (pRb). The homologous SV40 large T antigen protein has been studied thoroughly in recent decades, and has been associated with mesothelioma, osteosarcoma and brain tumours.However, the murine polyomavirus encodes for 154 additional amino acids that are rich in glycine and proline residues and could potentially play an important role towards cell transformation. Moreover, the polyoma virus protein has not been studied to this extent before, and structural and binding experiments conducted here reveal that it remains functional while natively unfolded. Nuclear Magnetic Resonance (NMR) spectroscopy was employed to characterise the protein's motional properties in its native state. A large part of the backbone residues was assigned, and regions interacting with pRb formed a localised structure. The determination of polyomavirus regions associated with retinoblastoma (PRAR) between residues 131 to 137 and to 181 have never been observed and represents a significant advance

Tetraspanin protein contributions to cancer

Among the 33 human tetraspanin proteins, CD151, CD9 and Tspan12 play particularly important roles in cancer. Tetraspanin CD151, in partnership with integrins α6β1 and α6β4, modulates tumour cell growth, invasion, migration, metastasis, signalling and drug sensitivity. Tetraspanin CD9 has suppressor functions in multiple tumour cell types. Major CD9 partner proteins, such as EWI-2 and EWI-F, may modulate these tumour-suppressor functions. Tetraspanin Tspan12 mutations are linked to a human disease called familial exudative vitreoretinopathy. In addition, as a regulator of the metalloprotease ADAM10 (a disintegrin and metalloprotease 10) maturation and function, Tspan12 probably contributes to the pro-tumorigenic functions of ADAM10.</jats:p

EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these “present-absent-present” TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is “absent-present-absent” in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 — molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients

Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells

Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)(2)D3 is unknown despite high levels of 1,25(OH)(2)D3 in decidua. Transcriptomic responses to 1,25(OH)(2)D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)(2)D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor beta (TGF beta). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)(2)D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)(2)D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)(2)D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.</p

Transmembrane Complexes of DAP12 Crystallized in Lipid Membranes Provide Insights into Control of Oligomerization in Immunoreceptor Assembly

The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces

Data from: Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals

The proposed mechanism for in meso crystallisation of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron Small Angle X-ray Scattering with a micro-sized X-ray beam. Crystal growth was found to occur from the Gyroid cubic mesophase. For one in four crystals a highly-oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallisation. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso, from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals