Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process.

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.

Resistance to neutralization by antibodies targeting the coiled coil of fusion-active envelope is a common feature of retroviruses.

The human T-cell leukemia virus transmembrane glycoprotein (TM) is a typical class 1 membrane fusion protein and a subunit of the viral envelope glycoprotein complex. Following activation, the TM undergoes conformational transitions from a native nonfusogenic state to a fusion-active pre-hairpin intermediate that subsequently resolves to a compact trimer-of-hairpins or six-helix bundle. Disruption of these structural transitions inhibits membrane fusion and viral entry and validates TM as an anti-viral and vaccine target. To investigate the immunological properties of fusion-active TM, we have generated a panel of monoclonal antibodies that recognize the coiled-coil domain of the pre-hairpin intermediate. Antibody reactivity is highly sensitive to the conformation of the coiled coil as binding is dramatically reduced or lost on denatured antigen. Moreover, a unique group of antibodies are 100-1000-fold more reactive with the coiled coil than the trimer-of-hairpins form of TM. The antibodies recognize virally expressed envelope, and significantly, some selectively bind to envelope only under conditions that promote membrane fusion. Most importantly, many of the antibodies potently block six-helix bundle formation in vitro. Nevertheless, viral envelope was remarkably resistant to neutralization by antibodies directed to the coiled coil. The data imply that the coiled coil of viral envelope is poorly exposed to antibody during membrane fusion. We suggest that resistance to neutralization by antibodies directed to fusion-associated structures is a common property of retroviral TM and perhaps of other viral class I fusion proteins. These observations have significant implications for vaccine design.

An antibody that blocks human T-cell leukemia virus type 1 six-helix-bundle formation in vitro identified by a novel assay for inhibitors of envelope function.

Fusion of the viral and cellular membranes is a critical step in the infection of cells by the human T-cell leukemia virus type 1 (HTLV-1) and this process is catalysed by the viral envelope glycoproteins. During fusion, the transmembrane glycoprotein (TM) is thought to undergo a transition from a rod-like pre-hairpin conformation that is stabilized by a trimeric coiled coil to a more compact six-helix-bundle or trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that the pre-hairpin motif is a valid target for antiviral therapy. Here, a stable, trimeric TM derivative that mimics the coiled-coil structure of fusion-active TM has been used to develop a plate-based assay to identify reagents that interfere with the formation of the six-helix bundle. The assay discriminates effectively between strong, weak and inactive peptide inhibitors of membrane fusion and has been used to identify a monoclonal antibody (mAb) that disrupts six-helix-bundle formation efficiently in vitro. The mAb is reactive with the C-helical region of TM, indicating that this region of TM is immunogenic. However, the mAb failed to neutralize HTLV-1 envelope-mediated membrane fusion, suggesting that, on native viral envelope, the epitope recognized by the mAb is obscured during fusion. This novel mAb will be of value in the immunological characterization of fusion-active structures of HTLV-1 TM. Moreover, the assay developed here will aid the search for therapeutic antibodies, peptides and small-molecule inhibitors targeting envelope and the HTLV-1 entry process.