Biology subjects

Slavoff, S. A.

Publications and source records attributed to Slavoff, S. A..

8 recordsLinked to original sources

bioRxiv2026

HaloPROTAC-mediated degradation reveals SOX10-dependent gene expression changes in melanoma cells

The transcription factor SOX10 is required for melanoma cell proliferation. However, SOX10 expression in single cells is heterogeneous, and low SOX10-expressing cells exhibit a dormant-invasive phenotype associated with drug resistance and metastasis. Despite longstanding interest in SOX10 as a diagnostic marker and potential therapeutic target in melanoma, the molecular mechanism and targets of SOX10 remain incompletely understood. New tools for the study and perturbation of SOX10 in cancer-relevant systems are needed to address these gaps in our knowledge. In this study, we engineered a patient-derived WW165 melanoma cell line for programmable SOX10 targeted proteolysis. We achieved this by replacing the endogenous copies of SOX10 with a SOX10-HaloTag transgene that supports cell growth, but can be rapidly degraded by the ubiquitin-proteasome system upon with a commercially available HaloPROTAC3 degrader molecule. We demonstrate that inducible degradation of SOX10-HaloTag faithfully recapitulates the known SOX10 transcriptional program, while also enabling discovery of SOX10 target genes previously undetected with standard genetic silencing approaches. We validated NAV2 and NCKAP5 as novel genes that are directly bound by SOX10 and coregulated with SOX10 expression in a patient cohort. This study demonstrates that new biological insights into the function of SOX10 can be enabled by targeted degradation and suggests that analogous approaches could prove powerful in the study of other transcription factors. We further suggest that the SOX10-HaloTag expressing melanoma cell line developed in this study could find further applications in investigating the consequences of SOX10 targeted degradation in vitro and in vivo.

biochemistry↗

bioRxiv2026

aaRSID, an engineered pyrrolysyl-tRNA synthetase platform for multi-probe proximity proteomics

Proximity labeling (PL) methods utilize spatially targeted chemical or enzymatic generation of a diffusible, reactive intermediate to covalently tag neighboring proteins in living systems. Unlike other tools for studying molecular interactions, PL can detect transient protein relationships with high spatial and temporal sensitivity, allowing for insight into their roles in biological processes. However, current enzymatic PL tools, such as TurboID and APEX2, are limited by their substrate structure and chemistry, which can generate significant background and/or perturb cellular physiology. To address these limitations, we have developed aminoacyl-tRNA synthetase ID (aaRSID), a PL tool that leverages an engineered pyrrolysyl tRNA synthetase (PylRS) for proximity labeling of proteins. We chose PylRS because it can catalyze promiscuous lysine labeling in the absence of its cognate tRNA and utilize a variety of non-canonical amino acids (ncAAs) as substrates. Here, we demonstrate aaRSID's intrinsic proximity labeling activity, use directed evolution to improve this activity, and apply the improved mutant (aaRSID-Ma1.3) for subcellular proteomics and multiplexed imaging. Our work establishes aminoacyl-tRNA synthetases as a new PL enzyme class and introduces a versatile chemical platform for developing ncAA-derived probes to map cellular microenvironments, greatly expanding the applications possible of PL technology.

biochemistry↗

bioRxiv2025

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)- based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.

genomics↗

bioRxiv2024

Alternative transcripts recode human genes to express overlapping, frameshifted microproteins

Overlapping genes were thought to be essentially absent from the human genome until the discovery of hundreds of translated, frameshifted internal open reading frames (iORFs) within annotated protein coding sequences (CDS). This would suggest that some human genes encode two completely different proteins; however, it is unclear how iORF-encoded proteins are translated, and whether they are broadly functional. We demonstrate that "non-coding" alternative transcripts lacking a complete protein coding sequence (CDS) are required for iORF translation. We also demonstrate that iORFs such as the conserved and antiapoptotic DEDD2 iORF encode proteins with functions distinct from the annotated proteins they overlap. This work thus provides a molecular and functional basis for dual coding of overlapping ORFs in human genes.

biochemistry↗

bioRxiv2024

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

molecular biology↗

bioRxiv2024

Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock

Hundreds of bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have recently been identified. Biological functions have been ascribed to an increasing number of microproteins, some of which play roles in bacterial stress responses. In this work, we provide evidence that GndA, a frameshifted 36-amino acid microprotein encoded in an internal open reading frame overlapping the 6-phosphogluconate dehydrogenase (6PGD) coding sequence (CDS), functions in the response to heat shock. We demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA is hydrophobic and associates with the pyruvate dehydrogenase complex and respiratory complex I (RCI). Consistent with a role in cellular homeostasis, GndA mutations alter the transcriptional responses to heat shock. These results demonstrate that GndA promotes bacterial cell growth at high temperature via a mechanism distinct from that of the 6PGD protein that it overlaps. GndA thus expands the paradigm of bacterial microproteins that function in cellular stress responses.

biochemistry↗

bioRxiv2021

The human RBM10 gene dually encodes a repressor of ribosome biogenesis that downregulates cell proliferation

Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing active or stress-induced synthesis could play important cellular roles, here, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated unannotated alt-proteins, one of which is upregulated after DNA damage stress. We further defined MINAS-60 (MIcroprotein that Negatively regulates ASsembly of the pre-60S ribosomal subunit), a nucleolar localized alt-protein co-encoded with human RBM10.Depletion of MINAS-60 increases the amount of the mature 60S ribosomal subunit, consequently upregulating global protein synthesis and cell proliferation by repressing late-stage pre-60S assembly and export of the 60S ribosome subunit to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of ribosome biogenesis, and demonstrate that chemoproteomics can enable generation of functional hypotheses for uncharacterized alt-proteins.

molecular biology↗

bioRxiv2020

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24

Thousands of previously unannotated small and alternative open reading frames (alt-ORFs) have recently been revealed in the human genome, and hundreds are now known to be required for cell proliferation. Many alt-ORFs are co-encoded with proteins of known function in multicistronic human genes, but the functions of only a handful are currently known in molecular detail. Using a proteomic strategy for discovery of unannotated short open reading frames in human cells, we report the detection of alt-RPL36, a 148-amino acid protein co-encoded with and overlapping human RPL36 (ribosomal protein L36). Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 in HEK 293T cells increases PI(4,5)P2 levels in the plasma membrane, upregulates the PI3K-AKT-mTOR signaling pathway, and increases cell size. Four serine residues of alt-RPL36 are phosphorylated, and mutation of these four serines to alanine abolishes interaction with TMEM24 and, consequently, abolishes alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as a novel regulator of PI(4,5)P2 synthesis upstream of the PI3K-AKT-mTOR signaling pathway. More broadly, these results show that the alt-RPL36 transcript can express two sequence-independent polypeptides from overlapping ORFs that regulate the same process - protein synthesis - via different molecular mechanisms (PI3K signaling and ribosome composition), expanding our knowledge of the mechanisms by which multicistronic human genes function.

genomics↗