AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins

Abstract

Summary Protein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, gel-based separation of modified and unmodified proteins, and an activity assay, we determine that the modified, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a molecular rationale behind lysosomal storage diseases and neurodegeneration.

Transforming Chemical Proteomics Enrichment into a High-Throughput Method Using an SP2E Workflow

“…The utilization of these probes has provided valuable insights into protein acetylation, palmitoylation, myristoylation, prenylation, glycosylation, ADP-ribosylation, and AMPylation. 1 , 12 − 14 , 22 − 25 In general, different chemical proteomic workflows follow the same sequence of key steps ( Figure 1 A). First, cultured cells are treated with the probe, which infiltrates the cellular system and competes with the endogenous substrate for the active site of the PTM writer enzymes.…”

“… 15 , 26 Following the click chemistry, proteins are precipitated to remove the excess biotin reagents and nonprotein components of the cell lysate. 1 , 13 In the next step, biotin-labeled proteins are enriched using avidin-coated beads. The critical part of this step is to maximize the efficiency of the washing to remove nonspecifically bound proteins and thus to reduce the complexity of the final MS sample while improving ratios between control- and probe-treated samples.…”

“…Protein post-translational modifications (PTMs) are crucial for the regulation and fine-tuning of many important biological processes such as neurodevelopment, − circadian clocks, aging, and impairment in numerous diseases. − The incredible diversity of genetic polymorphism, RNA splice variants, and PTMs results in many proteoforms, ,, which exceed the ∼20,000 human genes by approximately 50 times. This biological network orchestrates the most complex processes including brain development and ensures a dynamic response of the cells to an external stimulus.…”

“…Mass spectrometry (MS)-based chemical proteomics has allowed to reliably map protein PTMs across various experimental conditions. − A widespread application of the chemical proteomic strategy was enabled by parallel improvements of liquid chromatography technologies, gains in speed, and sensitivity of mass spectrometers and bioinformatic pipelines for protein identification and quantification. − Nowadays, chemical proteomics is used to uncover the scope of protein PTMs in different cell types by the development of small-molecule probes, which mimic their natural counterparts. The utilization of these probes has provided valuable insights into protein acetylation, palmitoylation, myristoylation, prenylation, glycosylation, ADP-ribosylation, and AMPylation. ,− ,− In general, different chemical proteomic workflows follow the same sequence of key steps (Figure A). First, cultured cells are treated with the probe, which infiltrates the cellular system and competes with the endogenous substrate for the active site of the PTM writer enzymes.…”

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

“…To streamline this process, we leveraged from fast (4 days) differentiation of hiPSCs engineered with doxycycline-inducible Neurogenin-1 and -2 cassette (iNGNs). [17] To test the feasibility of our approach in iNGNs, they were treated with Tyr- O -Pro at four different time points during differentiation into mature neurons (Figure S6). Similar to SH-SY5Y cells, the fluorescent band at around 50 kDa likely corresponding to the tubulins was present (Figure S6).…”