Transmembrane Domain Interactions Research Articles

Transmembrane domain-driven PD-1 dimers mediate T cell inhibition.

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases.

Identifying Transmembrane Interactions in Receptor Protein Tyrosine Phosphatase Homodimerization and Heterodimerization.

Receptor protein tyrosine phosphatases (RPTPs) are one of the key regulators of receptor tyrosine kinases (RTKs) and therefore play a critical role in modulating signal transduction. While the structure-function relationship of RTKs has been widely studied, the mechanisms modulating the activity of RPTPs still need to be fully understood. On the other hand, homodimerization has been shown to antagonize RPTP catalytic activity and appears to be a general feature of the entire family. Conversely, their documented ability to physically interact with RTKs is integral to their negative regulation of RTKs, but there is a yet-to-be proposed common model. However, specific transmembrane (TM) domain interactions and residues have been shown to be essential in regulating RPTP homodimerization, interactions with RTK substrates, and activity. Therefore, elucidating the contribution of the TM domains in RPTP regulation can provide significant insights into how these receptors function, interact, and eventually be modulated. This chapter describes the dominant-negative AraC-based transcriptional reporter (DN-AraTM) assay to identify specific TM interactions essential to homodimerization and heteroassociation with other membrane receptors, such as RTKs.

Structure of the priming arabinosyltransferase AftA required for AG biosynthesis of Mycobacterium tuberculosis

Arabinogalactan (AG) is an essential cell wall component in mycobacterial species, including the deadly human pathogen Mycobacterium tuberculosis. It plays a pivotal role in forming the rigid mycolyl-AG-peptidoglycan core for in vitro growth. AftA is a membrane-bound arabinosyltransferase and a key enzyme involved in AG biosynthesis which bridges the assembly of the arabinan chain to the galactan chain. It is known that AftA catalyzes the transfer of the first arabinofuranosyl residue from the donor decaprenyl-monophosphoryl-arabinose to the mature galactan chain (i.e., priming); however, the priming mechanism remains elusive. Herein, we report the cryo-EM structure of Mtb AftA. The detergent-embedded AftA assembles as a dimer with an interface maintained by both the transmembrane domain (TMD) and the soluble C-terminal domain (CTD) in the periplasm. The structure shows a conserved glycosyltransferase-C fold and two cavities converging at the active site. A metal ion participates in the interaction of TMD and CTD of each AftA molecule. Structural analyses combined with functional mutagenesis suggests a priming mechanism catalyzed by AftA in Mtb AG biosynthesis. Our data further provide a unique perspective into anti-TB drug discovery.

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane ‘accessory’ proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.

Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

Abstract 2524: Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay

Abstract In intrinsic apoptosis, the interaction network of the Bcl-2 protein family controls the decision over life and death. Cells are sentenced to death when pro-apoptotic multidomain effector proteins BAX, BAK or BOK oligomerize and form pores in the mitochondrial outer membrane. This releases cytochrome c, which induces the activation of cell-wrecking proteases, the caspases. Interactions of Bcl-2 proteins with other family members essentially regulate cell death. The interaction via the BH3 region was intensively studied in the last decades. As a result, small-molecule drugs, BH3-mimetics, were developed which bind and inhibit anti-apoptotic Bcl-2 proteins. Since anti-apoptotic Bcl-2 proteins are often overexpressed in hematopoietic malignancies, BH3-mimetics e.g. Venetoclax are approved for anti-cancer therapy. Unlike the BH3 interaction site, the c-terminal α9-helix referred to as the transmembrane domain (TMD) is mostly neglected in Bcl-2 interaction studies. However, TMDs not only dictate subcellular localization, but also substantially influence protein-protein interactions. Interestingly, Bcl-2 TMDs can harbor several tumor-specific mutations. The functional basis for TMD interaction as well as the resulting functional relevance for apoptosis signaling, however, remains poorly understood. To unravel the Bcl-2 TMD interaction network, we developed a split luciferase assay system enabling us to detect Bcl-2 TMD interactions in living cells. Simultaneously encoding for the expression of a fluorophore and TMD fusion peptides this system was used to generate fluorescence-normalized luminescence-based interaction data. Here, we identified a homotypic interaction pattern among effector TMDs of BAX, BAK and BOK. Molecular modelling of effector TMD interaction in mimics of cellular membranes also supports these findings. TMD swap experiments show significant influence of TMD sequence on subcellular localization and cell death signaling as assessed via confocal laser scanning microscopy and flow cytometry-based cell death assays. Moreover, we tested previously described mutations of the BAX-TMD (S184A, S184D) as well as a tumor-specific mutation (V180G) in the novel interaction assay. S184 (de-)phosphorylation as mimicked with S184A/S184D affects subcellular localization. In accordance, we find that S184A enhanced and S184D abolished interaction with wildtype BAX-TMD. Intriguingly, V180G not only modulates BAX subcellular localization but also prevents interaction with wildtype BAX-TMD. These findings verify a crucial role of Bcl-2 TMDs in subcellular localization and furthermore strongly support a function in interaction and cell death regulation. Further efforts to explore the Bcl-2 TMD interaction network as well as functional analysis of tumor-specific TMD mutations could pave the way to establish TMDs as a target of cancer therapy. Citation Format: Tobias B. Beigl, Alexander Paul, Sandra Weller, Benjamin Schäfer, Walter E. Aulitzky, Hans-Georg Kopp, Thomas Fellmeth, Kristyna Pluhackova, Markus Rehm, Frank Essmann. Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2524.

Computational design of BclxL inhibitors that target transmembrane domain interactions

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains (TMDs) have not been examined thoroughly despite their importance. Here, we developed a computational approach to design sequences that specifically modulate protein-protein interactions in the membrane. To illustrate this method, we demonstrated that BclxL can interact with other members of the B cell lymphoma 2 (Bcl2) family through the TMD and that these interactions are required for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the TMD of BclxL. Hence, we were able to prevent BclxL intramembrane interactions and cancel its antiapoptotic effect. These results advance our understanding of protein-protein interactions in membranes and provide a means to modulate them. Moreover, the success of our approach may trigger the development of a generation of inhibitors targeting interactions between TMDs.

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD.

The receptor protein tyrosine phosphatase (RPTP) PTPRJ (also known as DEP-1) has been identified as a negative regulator of the receptor tyrosine kinase FLT3 signalling in vitro. The inactivation of the PTPRJ gene in mice expressing the constitutively active, oncogenic receptor tyrosine kinase FLT3 ITD aggravated known features of leukaemogenesis, revealing PTPRJ's antagonistic role. FLT3 ITD mutations resulting in constitutively kinase activity and cell transformation frequently occur in patients with acute myeloid leukaemia (AML). Thus, in situ activation of PTPRJ could be used to abrogate oncogenic FLT3 signalling. The activity of PTPRJ is suppressed by homodimerization, which is mediated by transmembrane domain (TMD) interactions. Specific Glycine-to-Leucine mutations in the TMD disrupt oligomerization and inhibit the Epidermal Growth Factor Receptor (EGFR) and EGFR-driven cancer cell phenotypes. To study the effects of PTPRJ TMD mutant proteins on FLT3 ITD activity in cell lines, endogenous PTPRJ was inactivated and replaced by stable expression of PTPRJ TMD mutants. Autophosphorylation of wild-type and ITD-mutated FLT3 was diminished in AML cell lines expressing the PTPRJ TMD mutants compared to wild-type-expressing cells. This was accompanied by reduced FLT3-mediated global protein tyrosine phosphorylation and downstream signalling. Further, PTPRJ TMD mutant proteins impaired the proliferation and in vitro transformation of leukemic cells. Although PTPRJ's TMD mutant proteins showed impaired self-association, the specific phosphatase activity of immunoprecipitated proteins remained unchanged. In conclusion, this study demonstrates that the destabilization of PTPRJ TMD-mediated self-association increases the activity of PTPRJ in situ and impairs FLT3 activity and FLT3-driven cell phenotypes of AML cells. Thus, disrupting the oligomerization of PTPRJ in situ could prove a valuable therapeutic strategy to restrict oncogenic FLT3 activity in leukemic cells.

Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide

Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl− across cell membranes. NKCC1 mediates trans-epithelial Cl− secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.

Structural basis of tethered agonism of the adhesion GPCRs ADGRD1 and ADGRF1

Adhesion G protein-coupled receptors (aGPCRs) are essential for a variety of physiological processes such as immune responses, organ development, cellular communication, proliferation and homeostasis1–7. An intrinsic manner of activation that involves a tethered agonist in the N-terminal region of the receptor has been proposed for the aGPCRs8,9, but its molecular mechanism remains elusive. Here we report the G protein-bound structures of ADGRD1 and ADGRF1, which exhibit many unique features with regard to the tethered agonism. The stalk region that proceeds the first transmembrane helix acts as the tethered agonist by forming extensive interactions with the transmembrane domain; these interactions are mostly conserved in ADGRD1 and ADGRF1, suggesting that a common stalk–transmembrane domain interaction pattern is shared by members of the aGPCR family. A similar stalk binding mode is observed in the structure of autoproteolysis-deficient ADGRF1, supporting a cleavage-independent manner of receptor activation. The stalk-induced activation is facilitated by a cascade of inter-helix interaction cores that are conserved in positions but show sequence variability in these two aGPCRs. Furthermore, the intracellular region of ADGRF1 contains a specific lipid-binding site, which proves to be functionally important and may serve as the recognition site for the previously discovered endogenous ADGRF1 ligand synaptamide. These findings highlight the diversity and complexity of the signal transduction mechanisms of the aGPCRs.

Characterization of interactions within the Igα/Igβ transmembrane domains of the human B-cell receptor provides insights into receptor assembly

The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin and the Igα/β heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders, yet, despite the clinical significance of this protein complex, key regions (i.e., the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear and has been variously described by the mutually exclusive cross-linking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Igα and Igβ TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igβ TMD and strong self-association of the Igα TMD, which scanning mutagenesis revealed was entirely stabilized by an E–X10–P motif. We also demonstrated strong heterotypic interactions between the Igα and Igβ TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self-interactions and heterotypic interactions of the Igα TMD. Taken together, these results demonstrate that the TMDs of the human BCR are sites of strong protein–protein interactions that may direct BCR assembly, endoplasmic reticulum retention, and immune signaling.

A new class of 5-HT2A /5-HT2C receptor inverse agonists: Synthesis, molecular modeling, in vitro and in vivo pharmacology of novel 2-aminotetralins.

The 5-HT receptor subtypes 5-HT2A and 5-HT2C are important neurotherapeutic targets, though, obtaining selectivity over 5-HT2B and H1 receptors is challenging. Here, we delineated molecular determinants of selective binding to 5-HT2A and 5-HT2C receptors for novel 4-phenyl-2-dimethylaminotetralins (4-PATs). We synthesized 42 novel 4-PATs with halogen or aryl moieties at the C(4)-phenyl meta-position. Affinity, function, molecular modeling and 5-HT2A receptor mutagenesis studies were performed to understand structure-activity relationships at 5-HT2 -type and H1 receptors. Lead 4-PAT-type 5-HT2A /5-HT2C receptor inverse agonists were compared with pimavanserin, a selective 5-HT2A /5-HT2C receptor inverse agonist approved to treat Parkinson's disease-related psychosis, in the mouse head twitch response and locomotor activity assays, models relevant to antipsychotic drug development. Most 4-PAT diastereomers in the (2S,4R)-configuration bound non-selectively to 5-HT2A , 5-HT2C and H1 receptors, with >100-fold selectivity over 5-HT2B receptors, whereas diastereomers in the (2R,4R)-configuration bound preferentially to 5-HT2A over 5-HT2C receptors and had >100-fold selectivity over 5-HT2B and H1 receptors. Results suggest that G2385.42 and V2355.39 in 5-HT2A receptors (conserved in 5-HT2C receptors) are important for high affinity binding, whereas interactions with T1945.42 and W1584.56 determine H1 receptor affinity. The 4-PAT analog (2S,4R)-4-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, (2S,4R)-2k, a potent and selective 5-HT2A /5-HT2C receptor inverse agonist, had activity like pimavanserin in the mouse head twitch response assay but was distinct in not suppressing locomotor activity. The novel 4-PAT chemotype can yield selective 5-HT2A /5-HT2C receptor inverse agonists for antipsychotic drug development by optimizing ligand-receptor interactions in transmembrane domain 5. Chirality can be exploited to attain selectivity over H1 receptors, which may circumvent sedative effects.

Promoting receptor protein tyrosine phosphatase activity by targeting transmembrane domain interactions

Receptor protein tyrosine phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still-incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs.

Interactions of HIV gp41's membrane-proximal external region and transmembrane domain with phospholipid membranes from 31P NMR.

HIV-1 entry into cells requires coordinated changes of the conformation and dynamics of both the fusion protein, gp41, and the lipids in the cell membrane and virus envelope. Commonly proposed features of membrane deformation during fusion include high membrane curvature, lipid disorder, and membrane surface dehydration. The virus envelope and target cell membrane contain a diverse set of phospholipids and cholesterol. To dissect how different lipids interact with gp41 to contribute to membrane fusion, here we use 31P solid-state NMR spectroscopy to investigate the curvature, dynamics, and hydration of POPE, POPC and POPS membranes, with and without cholesterol, in the presence of a peptide comprising the membrane proximal external region (MPER) and transmembrane domain (TMD) of gp41. Static 31P NMR spectra indicate that the MPER-TMD induces strong negative Gaussian curvature (NGC) to the POPE membrane but little curvature to POPC and POPC:POPS membranes. The NGC manifests as an isotropic peak in the static NMR spectra, whose intensity increases with the peptide concentration. Cholesterol inhibits the NGC formation and stabilizes the lamellar phase. Relative intensities of magic-angle spinning 31P cross-polarization and direct-polarization spectra indicate that all three phospholipids become more mobile upon peptide binding. Finally, 2D 1H-31P correlation spectra show that the MPER-TMD enhances water 1H polarization transfer to the lipids, indicating that the membrane surfaces become more hydrated. These results suggest that POPE is an essential component of the high-curvature fusion site, and lipid dynamic disorder is a general feature of membrane restructuring during fusion.

Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.

The Role of the Membrane-Associated Domain of the Export Apparatus Protein, EscV (SctV), in the Activity of the Type III Secretion System.

Diarrheal diseases remain a major public health concern worldwide. Many of the causative bacterial pathogens that cause these diseases have a specialized protein complex, the type III secretion system (T3SS), which delivers effector proteins directly into host cells. These effectors manipulate host cell processes for the benefit of the infecting bacteria. The T3SS structure resembles a syringe anchored within the bacterial membrane, projecting toward the host cell membrane. The entry port of the T3SS substrates, called the export apparatus, is formed by five integral membrane proteins. Among the export apparatus proteins, EscV is the largest, and as it forms a nonamer, it constitutes the largest portion of the export apparatus complex. While there are considerable data on the soluble cytoplasmic domain of EscV, our knowledge of its membrane-associated section and its transmembrane domains (TMDs) is still very limited. In this study, using an isolated genetic reporter system, we found that TMD5 and TMD6 of EscV mediate strong self-oligomerization. Substituting these TMDs within the full-length protein with a random hydrophobic sequence resulted in a complete loss of function of the T3SS, further suggesting that the EscV TMD5 and TMD6 sequences have a functional role in addition to their structural role as membrane anchors. As we observed only mild reduction in the ability of the TMD-exchanged variants to integrate into the full or intermediate T3SS complexes, we concluded that EscV TMD5 and TMD6 are not crucial for the global assembly or stability of the T3SS complex but are rather involved in promoting the necessary TMD–TMD interactions within the complex and the overall TMD orientation to allow channel opening for the entry of T3SS substrates.

Abstract 1947: Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay

Abstract The intrinsic apoptosis signaling pathway comprises the intimate interaction network of the Bcl-2 protein family, in which both pro- and antiapoptotic members pushing cell fate to either survival or death. As a critical event, oligomerization of pore-forming effector proteins BAX, BAK and BOK drive mitochondrial outer membrane permeabilization (MOMP), which results in Cytochrome C release and ultimately apoptotic cell death. Thus far, investigations focus on protein-protein interactions mediated by the BH3-domain and hydrophobic groove. These interactions are targeted by BH3 mimetics that represent highly efficient anti-cancer drugs. In contrast, the C-terminal α-9 helices, known as transmembrane domains (TMD), tend to be neglected as a potential secondary interaction site with other Bcl-2 proteins. However, TMDs strongly affect subcellular localization and there is increasing evidence that TMDs crucially influence Bcl-2 protein interactions and therefore apoptosis signaling. Although TMDs are widely abundant in pro- and anti-apoptotic Bcl-2 proteins, their contribution to the complex Bcl-2 interaction network remains largely in the dark. Aiming to shed more light on the interactions of TMD among effector Bcl-2 proteins, we generated a bimolecular complementation assay in which effector TMD peptides are coupled to a bimolecular split-luciferase system. In addition, vectors encode for simultaneous expression of fluorophores to allow control of transgene expression. Human cell lines were co-transfected with combinations of TMD expression constructs yielding luminescence-based interaction data with a fluorescence-based normalization. Interestingly, we found an almost exclusively homotypic TMD interaction among the TMDs of BAX, BAK and BOK. Moreover, confocal microscopy with modified fluorophore-coupled TMD peptides revealed that TMD sequences were sufficient to locate BAX TMD and BAK TMD peptides effectively to mitochondria, while BOK TMD peptides were mainly localized to the endoplasmic reticulum. Strikingly, exchanging TMD sequences in full-length effector proteins profoundly modulated cell death induction. These results strongly suggest a central functional relevance of the α-9-helices in promoting homotypic interactions of effectors BAX, BAK and BOK during oligomerization and pore formation. In contrast, putative heterotypic interaction among effector proteins is not reflected by the newly generated TMD interaction assay. Thus, effector TMDs are a vital factor for specific protein localization and cell death induction displaying specific functionality in each effector protein. Our findings underline the importance of further investigating the TMD region as a potential oncogenic feature influencing interaction of Bcl-2 proteins and therefore as a potential target for therapeutic intervention. Citation Format: Tobias B. Beigl, Sandra Weller, Benjamin Schäfer, Kathrin Böpple, Lara-Sophie Rieder, Hans-Georg Kopp, Markus Rehm, Walter E. Aulitzky, Frank Essmann. Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1947.

Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation

The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75–TrkA receptor complex necessary for neuronal development stabilized by TMD interactions.

Promoting Receptor Protein Tyrosine Phosphatase Activity by Targeting Transmembrane Domain Interactions

Receptor Protein Tyrosine Phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still-incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs. We recently reported (10.1074/jbc.RA119.010229) that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ; also known as DEP1) is regulated by specific transmembrane (TM) residues, and that these interactions are essential in regulating its enzymatic activity and substrate access in cells. Building on these new insights and in response to the lack of selective agonists, we report here the design and testing of a tumor-selective peptide capable of binding to the TM domain of PTPRJ and disrupting its homodimerization. By doing so, it promotes PTPRJ TM-mediated access to EGFR, reduces the phosphorylation of EGFR (a known substrate) and other downstream signaling effectors, and antagonizes EGFR-driven cancer cell phenotypes. This peptide represents a novel allosteric and a possibly orthogonal way to target the activity of PTPRJ and RTKs phosphorylation. It could therefore be used not only as probes to tease out PTPRJ regulating mechanisms but also for therapeutic purposes via the attenuation of signaling by dysregulated RTKs in cancers. We also expect that the basic framework developed here can be extended to other RPTPs.

Acupuncture and Related Techniques for Pain Relief and Treatment of Heroin Addiction: Mechanisms and Clinical Application.

A neurochemical hypothesis of acupuncture analgesia suggests that the pain relief effect of acupuncture is primarily due to activation of a central endorphin system. It has been shown that the primary afferent sensory fibers, a mesolymbic neural circuit, and a descending inhibitory pathway are critical in acupuncture analgesia. The therapeutic effects of electroacupuncture (EA) and related techniques, such as transcutaneous electroacupoint stimulation (TEAS), are frequency-dependent: different frequencies of EA activate different brain regions and release different neuropeptides. EA and TEAS have been used successfully to treat heroin addiction. Activation of endorphin gene expression and release by TEAS can explain the dramatic attenuation of withdrawal syndrome and prolongation of retention time during and after detoxification treatment in patients who are addicted to heroin. However, repeated EA at high intensity should be avoided because it can induce a gradual loss of the analgesic effect. Opioid-receptor desensitization occurs and is manifested as decreased ligand-binding affinity and second-messenger detachment. Repeated large doses of morphine induce morphine tolerance. Cross-tolerance between morphine and EA suggests similar underlying mechanisms. Recent studies have demonstrated that excessive activation of cholecystokinin (CCK), an antiopioid peptide, appears to be responsible. CCK-receptor subtype B (CCKBR) and opioid μ-receptor are co-expressed in the dorsal-horn neurons. Activation of CCKBR promotes formation of heteromerization of morphine-receptor and CCKBR. Interaction of the third transmembrane domain between the 2 receptors resulted in the reduced binding affinity of the opioid receptor.