Diving into the hidden world of AltProts: unraveling functions and interactions

For several years, we have focused on identifying an extended proteome beyond the traditionally referenced proteins (RefProt) contained in databases. While most proteomic studies concentrate on these known proteins, there is another layer to the proteome called the "non-referenced or Ghost proteome," which includes "Alternative proteins" (AltProt). These proteins originate from regions typically annotated as non-coding in transcripts, such as the 5' and 3' untranslated regions (UTRs), alternative reading frames (+1 or +2), and non-coding RNAs (ncRNAs, lncRNAs, circRNAs). Because AltProts are not included in standard databases, they are difficult to detect, and their functions are not well understood. Our research aims to uncover these proteins and elucidate their roles in cellular processes.

This raises the critical question: "How can we determine the function of AltProts given their lack of molecular tools?" To address this, we are developing innovative strategies to identify and characterize the functions of AltProts. By leveraging advanced techniques such as crosslinking mass spectrometry (XL-MS), we aim to reveal protein-protein interactions involving AltProts, thereby predicting their roles in various cellular pathways and processes. This approach has already shown promise, as we have identified interactions between AltProts and known proteins, providing insights into their potential functions and contributions to cellular mechanisms.

Using the XL-MS approach, we can identify protein-protein interactions in an untargeted manner. This technique allows us to reveal the presence of AltProts within protein interaction networks and predict their roles in signaling pathways. For instance, using this approach, IP_166911.1 (AltATAD2) was found interacting with RPL10 and AUF1, possibly regulating the binding of 5S ribosomal RNA in the ribosome. Similarly, in glioblastoma cells (NCH82), several AltProts were identified interacting with TPM4, and some of these interactions were confirmed by Immunofluorescence and TurboID. We have optimized the detection of crosslinked peptides through subcellular fractionation techniques, enabling the identification of AltProts specific to cellular localization and their interactions. This approach revealed AltProts interacting with HLA-B, particularly at the B2M interaction site. AltProt IP_2284785 might be involved in the stability or inhibition of the MHC-I complex.

Our ongoing research aims to expand the understanding of AltProts and their roles in cellular processes, potentially leading to new insights into cellular mechanisms and the development of novel therapeutic targets.