The eukaryotic 26S proteasome is a large multi-subunit complex that degrades the majority of proteins in the cell under normal conditions. The 26S proteasome can be divided into two subcomplexes: the 19S regulatory particle (RP) and the 20S core particle (CP). Most substrates are first covalently modified by ubiquitin, which then directs them to the proteasome. The function of the RP is to recognize, unfold, deubiquitylate and translocate substrates into the CP, which contains the proteolytic sites of the proteasome. Given the abundance and subunit complexity of the proteasome, the assembly of this ~2.5 MDa complex must be carefully orchestrated to ensure its correct formation. In recent years, significant advances have been made in the understanding of proteasome assembly, structure and function. Technical advances in cryo-electron microscopy have resulted in a series of atomic cryo-EM structures of both human and yeast 26S proteasomes. These structures have illuminated new intricacies and dynamics of the proteasome. In this review, we focus on the mechanisms of proteasome assembly, particularly in light of recent structural information.

Proteasome assembly utilizes multiple dedicated assembly chaperones and is regulated by signaling pathways that respond to diverse stress conditions. To discover new factors influencing proteasome base assembly, we screened a tiled high-copy yeast genomic library to identify dosage suppressors of a temperature-sensitive proteasome regulatory particle (RP) base mutant. The screen identified Nst1, a protein that when overexpressed, specifically suppressed the temperature sensitivity and proteasome-assembly defects of multiple base mutants. Nst1 overexpression reduced cytosolic RP ATPase (Rpt) aggregates in nas6Δ rpn14Δ cells, which lack two RP assembly chaperones. Nst1 is highly polar and predicted to have numerous intrinsically disordered regions, characteristics commonly found in proteins that can segregate into membraneless condensates. In agreement with this, both endogenous and overexpressed Nst1 could form cytosolic puncta that co-localized with processing body (P-body) components. Consistent with the accumulation of translationally inactive mRNAs in P-bodies, Nst1 overexpression inhibited global protein translation in nas6Δ rpn14Δ cells. Translational inhibition is known to suppress aggregation and proteasome assembly defects in base mutants under heat stress . Our data indicate that Nst1 is a previously overlooked P-body component that when expressed at elevated levels inhibits translation, prevents Rpt subunit aggregation, and rescues proteasome assembly under stress conditions.

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Introduction: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is expressed on the surface of some tumor epithelial cells and is a potential therapeutic target for antibody-drug conjugates such as tusamitamab ravtansine in development. An IHC assay has been fit-for-purpose validated in non-small cell lung cancer (NSCLC) for evaluation of clinical samples using a proprietary CEACAM5-specific murine antibody (Sanofi clone 769) and the Dako/Agilent Autostainer Link 48 platform. Here we compare 5 commercial antibody clones to the validated IHC assay for CEACAM5. Methods: We tested 5 commercial clones (CI-P83-1 [Santa Cruz Biotechnology], 487609 [R&D Systems], OTI1D4 [Thermo Fisher], EPCEAR7 [Abcam], and 327 [Sino Biological]) to evaluate CEACAM5 detection in formalin-fixed, paraffin-embedded (FFPE) human cancer tissues from Discovery Life Sciences using the Leica BOND III platform. The staining pattern and intensity of CEACAM5 reactivity were compared for clone 769 and the commercial clones in the same FFPE samples. Initial IHC testing used gastric cancer tissues with continued assay development in colorectal cancer tissues. Commercially available (HT-29, MKN-45, BxPC-3, and PC-3) cell lines and CHO cells overexpressing other CEACAM targets (Sanofi) were used to test for cross reactivity and specificity. IHC staining in cell line controls and human cancer tissues (small cell lung cancer, NSCLC adenocarcinoma, squamous cell NSCLC, and pancreatic cancer), was performed to further evaluate staining accuracy to CEACAM5 and to better compare clones. Results: Of the 5 antibodies tested, 3 were deemed unsatisfactory due to diffuse cytoplasmic staining or overstaining and were not evaluated further. Clones CI-P83-1 and 487609 best matched the pattern and intensity of clone 769 staining and were evaluated at various dilutions in gastric and colorectal cancer samples to determine optimal antibody concentration and to demonstrate range and linearity of the IHC assays. Clones CI-P83-1 and 487609 accurately and specifically detected CEACAM5 without cross reactivity to similar targets in cell lines. In the human cancer tissues, clone CI-P83-1 tended to show greater parity to clone 769 than clone 487609, which sometimes showed slightly higher CEACAM5 signal than clone 769. Clone CI-P83-1 was appropriately reactive in CEACAM5 expression control cells, highly reactive in CHO cells overexpressing CEACAM5, and lacked cross reactivity in CHO cells overexpressing CEACAM types 1, 6, 7, or 8. A fit-for-purpose validation of clone CI-P83-1 in non-squamous NSCLC with clone 769 showed comparable staining in this indication. Conclusions: Among the 5 tested clones, there were differences in staining pattern, intensity, and cross reactivity. CI-P83-1 was the closest to 769 among the 5 clones. Further testing and validation are required to understand the finer differences between 769 and CI-P83-1. Citation Format: Chin Leng Cheng, Hongli Chen, Karen Kirchner, Gregory Cesarone. Characterization of a novel immunohistochemistry (IHC) assay for CEACAM5 using a commercial antibody [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 2763.