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Biological membrane fusion proceeds via an essential topological transition of the two membranes involved. Known players such as certain lipid species and fusion proteins are generally believed to alter the free energy and thus the rate of the fusion reaction. Quantifying these effects by theory poses a major challenge since the essential reaction intermediates are collective, diffusive and of a molecular length scale. We conducted molecular dynamics simulations in conjunction with a state-of-the-art string method to resolve the minimum free-energy path of the first fusion intermediate state, the so-called stalk. We demonstrate that the isolated transmembrane domains (TMDs) of fusion proteins such as SNARE molecules drastically lower the free energy of both the stalk barrier and metastable stalk, which is not trivially explained by molecular shape arguments. We relate this effect to the local thinning of the membrane (negative hydrophobic mismatch) imposed by the TMDs which favors the nearby presence of the highly bent stalk structure or prestalk dimple. The distance between the membranes is the most crucial determinant of the free energy of the stalk, whereas the free-energy barrier changes only slightly. Surprisingly, fusion enhancing lipids, i.e., lipids with a negative spontaneous curvature, such as PE lipids have little effect on the free energy of the stalk barrier, likely because of its single molecular nature. In contrast, the lipid shape plays a crucial role in overcoming the hydration repulsion between two membranes and thus rather lowers the total work required to form a stalk.

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG 4 ) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG 4 -HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.

Chimeric antigen receptor (CAR)-T cells have demonstrated significant clinical potential; however, their strong antitumor activity may cause severe adverse effects. To ensure efficacy and safe CAR-T cell therapy, it is important to understand CAR’s structure–activity relationship. To clarify the role of hinge and transmembrane domains in CAR and CAR-T cell function, we generated different chimeras and analyzed their expression levels and antigen-specific activity on CAR-T cells. First, we created a basic CAR with hinge, transmembrane, and signal transduction domains derived from CD3ζ, then we generated six CAR variants whose hinge or hinge/transmembrane domains originated from CD4, CD8α, and CD28. CAR expression level and stability on the T cell were greatly affected by transmembrane rather than hinge domain. Antigen-specific functions of most CAR-T cells depended on their CAR expression levels. However, CARs with a CD8α- or CD28-derived hinge domain showed significant differences in CAR-T cell function, despite their equal expression levels. These results suggest that CAR signaling intensity into T cells was affected not only by CAR expression level, but also by the hinge domain. Our discoveries indicate that the hinge domain regulates the CAR signaling threshold and the transmembrane domain regulates the amount of CAR signaling via control of CAR expression level.

SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A’s JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A’s JMD; and (3) palmitoylation deficiency mutations in STX1A’s TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca 2+ -evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A’s JMD regulates the palmitoylation of STX1A’s TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A’s JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.

Initiation of host cell infection by an enveloped virus requires a viral-to-host cell membrane fusion event. This event is mediated by at least one viral transmembrane glycoprotein, termed the fusion protein, which is a key therapeutic target. Viral fusion proteins have been studied for decades, and numerous critical insights into their function have been elucidated. However, the transmembrane region remains one of the most poorly understood facets of these proteins. In the past ten years, the field has made significant advances in understanding the role of the membrane-spanning region of viral fusion proteins. We summarize developments made in the past decade that have contributed to the understanding of the transmembrane region of viral fusion proteins, highlighting not only their critical role in the membrane fusion process, but further demonstrating their involvement in several aspects of the viral lifecycle.

Transmembrane domain-containing 7 (CMTM7) is a protein located at the plasma membrane. It plays a role in regulating the development and immune microenvironment of tumor cells. However, the impact of CMTM7 on hepatocellular carcinoma (HCC) is not well understood. To better understand the role of CMTM7 in HCC, the correlations of CMTM7 with clinical characteristics, patient prognosis, chronic inflammation, and immune cell infiltration were analyzed using tissue microarray slides, sequencing datasets and various analysis tools (Web). The bulk sequencing analysis indicated that elevated expression of CMTM7 appears to promote chronic inflammation, immunosuppression, M2 macrophage infiltration, a diminished response to cancer immunotherapy, and an unfavorable clinical prognosis in patients with hepatocellular carcinoma (HCC). Further investigation through single-cell RNA sequencing and multiple fluorescence staining demonstrated that CMTM7 serves as a molecular marker for M2 macrophages and is associated with T cell exhaustion as well as highly plastic stem-like characteristics. We propose that CMTM7 may represent a novel immune checkpoint for HCC patients experiencing suboptimal therapeutic outcomes. Utilizing the Connectivity Map and AutoDock Vina, we predicted two potential compounds targeting CMTM7—fasudil and arachidonyltrifluoromethane—as promising therapeutic candidates. Collectively, these findings suggest that CMTM7-positive macrophages play significant roles in establishing an immunosuppressive tumor microenvironment while promoting highly plastic and stem-like traits in HCC cells, ultimately contributing to poor prognostic outcomes.

Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has proven effective for treating relapsed or refractory acute B cell leukemia. However, challenges such as cytokine release syndrome, T cell dysfunction, and exhaustion persist. Enhancing CAR-T cell efficacy through changing CAR internalization and recycling is a promising approach. The transmembrane domain is the easiest motif to optimize for modulating CAR internalization and recycling without introducing additional domains, and its impact on CAR internalization and recycling has not yet been thoroughly explored. In this study, we aim to enhance CAR-T cell function by focusing on the solely transmembrane domain design. Utilizing plasmid construction and lentivirus generation, we get two different transmembrane CAR-T cells [19CAR-T(1a) and 19CAR-T(8α)]. Through co-culture with tumor cells, we evaluate CAR dynamic change, activation levels, exhaustion markers, mitochondrial function, and differentiation in both CAR-T cells. Furthermore, immunofluorescence microscopy analysis is performed to reveal the localization of internalized CAR molecules. RNA sequencing is used to detect the transcriptome of activated CAR-T cells. Finally, a mouse study is utilized to verify the anti-tumor efficacy of 19CAR-T(1a) cells . Our findings demonstrate that 19CAR-T(1a) has lower surface CAR expression, faster internalization, and a higher recycling rate compared to 19CAR-T(8α). Internalized 19CAR(1a) co-localizes more with early and recycling endosomes, and less with lysosomes than 19CAR(8α). These features result in lower activation levels, less cytokine release, and reduced exhaustion markers in 19CAR-T(1a). Furthermore, CAR-T cells with CD1a transmembrane domain also exhibit a superior anti-tumor ability and reduced exhaustion . Overall, we demonstrate that the transmembrane domain plays a critical role in CAR-T cell function. An optimized transmembrane domain can alleviate cytokine release syndrome and reduce CAR-T cell exhaustion, providing a direction for CAR design to enhance CAR-T cell function.

Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.

Previous works have shown the existence of protein partnership, belonging to a MultiStep Phosphorelay (MSP), potentially involved in osmosensing in Populus. The first actor of this signalling pathway belongs to the histidine-aspartate kinase (HK) family, which also includes the yeast osmosensor Sln1, as well as the Arabidopsis putative osmosensor AHK1. In poplar, the homologous AHK1 protein corresponds to a pair of paralogous proteins, HK1a and HK1b, exhibiting an extracellular domain (ECD), as in Sln1 and AHK1. An ECD alignment of AHK1-like proteins, from different plant species, showed a particularly well conserved ECD and revealed the presence of a cache domain. This level of conservation suggested a functional role of this domain in osmosensing. Thus, we tested this possibility by modelling assisted mutational analysis of the cache domain of the Populus HK1 proteins. The mutants were assessed for their ability to respond to different osmotic stress and the results point to an involvement of this domain in HK1 functionality. Furthermore, since HK1b was shown to respond better to stress than HK1a, these two receptors constituted a good system to search for osmosensing determinants responsible for this difference in efficiency. With domain swapping experiments, we finally demonstrated that the cache domain, as well as the second transmembrane domain, are involved in the osmosensing efficiency of these receptors.

The chimeric antigen receptor (CAR) is a synthetic and modular molecule composed of both signaling and non-signaling domains that allows a T cell to recognize cell surface antigens and trigger cytolytic functionality. It is appreciated that the non-signaling structural components, including the linker, hinge, and transmembrane domains, can dramatically alter how the CAR molecule interacts with itself and other endogenous molecules in the immune synapse. Herein, we describe the current understanding of how the structural domains can alter CAR T cell therapeutic efficacy and highlight how knowledge of the target antigen characteristics can inform CAR design choices.