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A light-resuming strategy as a screening method for Sec61 inhibitors down-modulating PD-L1 expression

ABSTRACT: The perturbation of protein translocation into the secretory pathway using Sec61 translocon inhibitors is a novel and promising strategy for tackling many pathological situations, including cancer and viral infections. However, a highly sensitive and direct screening platform for selecting Sec61 inhibitors is unavailable. Here, we develop a new “resuming luminescence upon translocation interference” (RELITE) assay capable of selecting Sec61 inhibitors in a single round of screening. This assay exploits the inactivation of firefly luciferase, once translocated into the endoplasmic reticulum (ER), and the possibility of diverting and “re-lighting” luciferase into the cytosol by a Sec61 inhibitor. Using this method, we selected small molecules capable of hampering the protein expression of the PD-L1 immune checkpoint by interfering with its ER translocation and delivering it for degradation. In conclusion, our screening method will greatly facilitate the selection of Sec61 inhibitors for down-modulating the expression of many disease-relevant proteins.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Massimo D'Agostino

LAB HEAD: Massimo D

PROVIDER: PXD063288 | Pride | 2025-08-04

REPOSITORIES: Pride

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			Action	DRS
	2025-01-31_MS_MDA_ProteomeGB138_BL458_02uM_01.raw	Raw
	2025-01-31_MS_MDA_ProteomeGB138_BL458_02uM_02.raw	Raw
	2025-01-31_MS_MDA_ProteomeGB138_BL458_02uM_03.raw	Raw
	2025-01-31_MS_MDA_ProteomeGB138_BL458_02uM_04.raw	Raw
	2025-01-31_MS_MDA_ProteomeGB138_BL458_20uM_01.raw	Raw

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Project description:Maintenance of cellular homeostasis and functionality requires rapid translocation of newly synthesized lipids across the membrane leaflets of the endoplasmic reticulum (ER) where majority of cellular lipid biosynthesis takes place. This process is facilitated without the need for ATP by specific membrane proteins—scramblases—a few of which have been very recently identified in the ER. We have previously resolved the structure of the translocon-associated protein (TRAP) bound to the Sec61 translocon, and found this complex to render the membrane locally thinner. We thus hypothesized that the translocon could provide scrambling pathways in the ER membrane. Here, we first observed non-selective lipid scrambling by reconstituted translocon complexes using complementary fluorescence approaches. Our experiments with inhibitors that block the putative Sec61 scrambling site suggest that an additional scrambling pathway is active in the translocon. Our extensive molecular dynamics simulations indicate that this additional pathway could be provided by the trimeric bundle of TRAP subunits. Its crevice rich in polar residues shields lipid head groups as they traverse the membrane via a credit card mechanism. We analyzed the kinetics and thermodynamics of lipid scrambling by both Sec61 and TRAP and demonstrated that local membrane thinning provides a key contribution to scrambling efficiency. Both proteins appear selective towards phosphatidylcholine lipids over phosphatidylethanolamine and phosphatidylserine, though this trend reflects the natural flip-flop tendencies of these lipids in a protein-free membrane. The identified scrambling pathway in the Sec61 translocon is located at the lateral gate region, which is likely either closed or occupied by a nascent polypeptide during protein translocation. Moreover, our simulations suggest Sec61 scrambling to be impeded by physiological salt concentration. Together with the observations with Sec61 inhibitors, this indicates the presence of an alternative scrambling site in the translocon complex, and the metazoan-specific transmembrane helix bundle of TRAPβ, TRAPγ, and TRAPδ seems like a viable candidate with scrambling activity that is insensitive to the translocon functional state and solvent conditions.

Project description:Glioblastoma multiforme (GBM) is the most prevalent type of adult brain tumor, and one of the deadliest tumors known to mankind. The genetic understanding of GBM is, however, limited, and the molecular mechanisms which facilitate GBM cell survival and growth within the tumor microenvironment are largely unknown. We applied digital karyotyping and single nucleotide polymorphism (SNP) arrays to screen for copy number changes in GBM samples and found that the most frequently amplified region is at chromosome 7p11.2. The high resolution of digital karyotyping and SNP arrays permits the precise delineation of amplicon boundaries and has enabled identification of the minimal region of amplification at 7p11.2, which contains two genes, EGFR and SEC61?. SEC61? encodes a subunit of a heterotrimeric protein channel located in the endoplasmic reticulum (ER). In addition to its high frequency of gene amplification in GBMs, SEC61? is also remarkably overexpressed in 77% of GBMs, but not in lower-grade gliomas. The siRNA-mediated knockdown of SEC61? expression in tumor cells led to growth suppression and apoptosis. Furthermore, we showed that pharmacological ER stress agents induce SEC61? expression in GBM cells. Together, these results indicate that aberrant expression of SEC61? serves significant roles in GBM cell survival, likely via a mechanism that is involved in the cytoprotective ER stress-adaptive response to the tumor microenvironment. hSETD1A was silenced in HCT116 cells using retrovirus harboring shRNA specifically against the hSETD1A gene. 10?g RNA was reverse transcribed to cDNA using the Applied Biosystems High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions (Applied Biosystems). cDNA products were treated with 100ng RNaseA and then purified using the Qiagen PCR purification kit according to the manufacturer’s instructions. NimbleGen Human Gene Expression array was purchased from Roche Applied Sciences. cDNA samples were labeled, Cy3 hybridized, and processed at the FSU NimbleGen Microarray Facility at Florida State University.

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A light-resuming strategy as a screening method for Sec61 inhibitors down-modulating PD-L1 expression

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