Chemical Proteomics Reveals Protein Tyrosination Extends Beyond the Alpha-Tubulins in Human Cells.

Tubulin detyrosination-tyrosination cycle regulates the stability of microtubules. With respect to α-tubulins, the tyrosination level is maintained by a single tubulin-tyrosine ligase (TTL). However, the precise dynamics and tubulin isoforms which undergo (de)tyrosination in neurons are unknown. Here, we exploit the substrate promiscuity of the TTL to introduce an O-propargyl-l-tyrosine to neuroblastoma cells and neurons. Mass spectrometry-based chemical proteomics in neuroblastoma cells using the O-propargyl-l-tyrosine probe revealed previously discussed tyrosination of TUBA4A, MAPRE1, and other non-tubulin proteins. This finding was further corroborated in differentiating neurons. Together we present the method for tubulin tyrosination profiling in living cells. Our results show that detyrosination-tyrosination is not restricted to α-tubulins with coded C-terminal tyrosine and is thus involved in fine-tuning of the tubulin and non-tubulin proteins during neuronal differentiation.

Drug-like Inhibitors of DC-SIGN Based on a Quinolone Scaffold.

DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) is a pattern recognition receptor expressed on immune cells and involved in the recognition of carbohydrate signatures present on various pathogens, including HIV, Ebola, and SARS-CoV-2. Therefore, developing inhibitors blocking the carbohydrate-binding site of DC-SIGN could generate a valuable tool to investigate the role of this receptor in several infectious diseases. Herein, we performed a fragment-based ligand design using 4-quinolone as a scaffold. We synthesized a library of 61 compounds, performed a screening against DC-SIGN using an STD reporter assay, and validated these data using protein-based 1H-15N HSQC NMR. Based on the structure-activity relationship data, we demonstrate that ethoxycarbonyl or dimethylaminocarbonyl in position 2 or 3 is favorable for the DC-SIGN binding activity, especially in combination with fluorine, ethoxycarbonyl, or dimethylaminocarbonyl in position 7 or 8. Furthermore, we demonstrate that these quinolones can allosterically modulate the carbohydrate binding site, which offers an alternative approach toward this challenging protein target.

Clickable report tags for identification of modified peptides by mass spectrometry.

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.