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Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared to their canonical counterparts. Such proteoforms can have altered localizations, stabilities and functions. While proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. Additionally, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared to their canonical counterpart, thus further expanding the functional diversity of proteomes.

SUMMARYMost eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.In BriefVarland, Silva et al. define that a major cellular role of N-terminal acetylation is shielding proteins from proteasomal degradation by specific ubiquitin ligases. The human N-terminal acetyltransferase NatC protects the neddylation regulator UBE2M from degradation, while overexpression of Drosophila UBE2M/UbcE2M rescues the longevity and motility defects of NatC deletion.HighlightsN-terminal acetylation by NatC protects proteins from degradation, including UBE2MUBR4-KCMF1 targets unacetylated N-terminal Met followed by a hydrophobic residueDrosophila NatC is required for adult longevity and motility in elderlyOverexpression of UBE2M/UbcE2M suppresses Drosophila NatC deletion phenotypes

Especially in eukaryotes, the N-terminal acetylation status of a protein reveals translation initiation sites and substrate specificities and activities of N-terminal acetyltransferases (NATs). Here, we discuss a bottom-up proteomics protocol for the enrichment of N-terminal peptides via strong cation exchange chromatography. This protocol is based on depleting internal tryptic peptides from proteome digests through their retention on strong cation exchangers, leaving N-terminally acetylated/blocked peptides enriched among the nonretained peptides. As such, one can identify novel N-terminal proteoforms and quantify the degree of N-terminal protein acetylation.

Ribosome profiling has revealed translation outside canonical coding sequences, including translation of short upstream ORFs, long noncoding RNAs, overlapping ORFs, ORFs in UTRs, or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling, and CRISPR-based screens showed that hundreds of ORFs derived from noncoding transcripts produce (micro)proteins, whereas other studies failed to find evidence for such types of noncanonical translation products. Here, we attempted to discover translation products from noncoding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. We show that, theoretically our strategy facilitates the detection of translation events of transcripts from non -coding regions but experimentally only find 19 peptides that might originate from such translation events. Finally, Virotrap-based interactome analysis of two N-terminal proteoforms originating from noncoding regions showed the functional potential of these novel proteins.

The N-terminus of a protein can encode several protein features, including its half-live and its localization. As the proteomics field remains dominated by bottom-up approaches and as N-terminal peptides only account for a fraction of all analyzable peptides, there is a need for their enrichment prior to analysis. COFRADIC, TAILS, and the subtiligase method were among the first N-terminomics methods developed, and several variants and novel methods were introduced that often reduce processing time and/or the amount of material required.We present an overview of how the field of N-terminomics developed, including a discussion of the founding methods, several updates made to these and introduce newer methods such as TMPP-labeling, biotin-based methods besides some necessary improvements in data analysis.N-terminomic methods remain being used and improved methods are published however, more efficient use of contemporary mass spectrometers, promising data-independent approaches, and mass spectrometry-free single peptide or protein sequences may threat the N-terminomics field.

Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins, while other studies failed to find evidence for such types of non-canonical translation products. Here, we attempted to discover translation products from non-coding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. Theoretically, we show that our strategy facilitates the detection of translation events of transcripts from non-coding regions, but experimentally only find 19 peptides (less than 1% of all identified peptides) that might originate from such translation events. Virotrap based interactome analysis of two N-terminal proteoforms originating from non-coding regions finally showed the functional potential of these novel proteins.

The collection of chemically different protein variants, or proteoforms, by far exceeds the number of protein-coding genes in the human genome. Major contributors are alternative splicing and protein modifications. In this review, we focus on those proteoforms that differ at their N termini with a molecular link to disease. We describe the main underlying mechanisms that give rise to such N-terminal proteoforms, these being splicing, initiation of protein translation, and protein modifications. Given their role in several human diseases, it is becoming increasingly clear that several of these N-terminal proteoforms may have potential as therapeutic interventions and/or for diagnosing and prognosing their associated disease.