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AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins

ABSTRACT: Protein AMPylation is a prevalent posttranslational modification with an emerging role in neurodevelopment and neurodegeneration. Although in metazoans the two highly conserved protein AMP-transferases together with diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques the function of this modification remains largely unknown. Particularly problematic is a localisation of thus far identified AMPylated proteins and putative AMP-transferases. Here, we uncover protein AMPylation as a novel lysosomal protein posttranslational modification characteristic for differentiating neurons. The AMPylated soluble form of exonuclease PLD3 localised in lysosomes shows dramatic increase during the differentiation of neuronal cell lineages. Similar AMPylation pattern has been observed for a lysosomal acid phosphatase ACP2. Our discovery was enabled by combination of chemical proteomics and novel gel-based separation technique of modified and non-modified proteins. Together, our findings expose further the connection between the protein AMPylation and neurodevelopment and reveal a novel lysosomal posttranslational modification.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

SUBMITTER: Tobias Becker

LAB HEAD: Dr. Pavel Kielkowski

PROVIDER: PXD023873 | Pride | 2021-12-01

REPOSITORIES: Pride

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			Action	DRS
	20200316_TB10_24_C1.raw	Raw
	20200316_TB10_24_C2.raw	Raw
	20200316_TB10_24_C3.raw	Raw
	20200316_TB10_24_C4.raw	Raw
	20200316_TB10_24_P1.raw	Raw

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Pronucleotide probe for in vivo imaging of protein AMPylation.

Project description:Protein AMPylation is a prevalent posttranslational modification in human cells involved in the regulation of unfolded protein response and neural development. Here we present a novel pro-N6azA probe suitable for in vivo imaging and chemical proteomics profilling of AMPylated proteins. The pro-N6azA probe provides stable fluorescent labelling in living cells accesible by straightforward strain-promoted azide-alkyne click reaction. Application of this probe may contribute significantly to the analysis of protein AMPylation during various metabolic processes including neurodevelopment and unfolded protein response.

2019-12-10 | PXD016011 | Pride

AMpylation is a hallmark of human neuronal differentiation

Project description:The AMPylation is novel protein post-translation modification. In consist of attachment of the adenosine monophosphate onto the serine, threonine or tyrosine amino acid residues of the protein. This process is catalysed by bacterial effectors or in human by protein FICD (also called HYPE) which contain conserved fic domain. In our study we have focused on identification of the unknown AMPylated proteins and their function in human cells (HeLa, A549, SH-SY5Y, iPSCs, NPCs and neurons) and cerebral organoids (COs). For this purpose we designed and synthesized the small molecule N6-propargyl phosphoramidate adenosine (PAK-76) which was used for in situ labelling of the AMPylation in cells and COs. Subsequnetly the the propargyl tag was employed for preparation of chemical-proteomic samples which were measured by LC-MS/MS. This approach confirmed the known AMPylation of heat shock protein HSPA5 and found diverse group of novel AMPylation targets. Several cathepsins found as AMPylation targets were further characterized by identification of AMP binding site. Based on our chemical proteomic data we found out that neurons and neuronal like cells contain distinct AMPylatino pattern from other studied cell types. This also suggested the importance of the AMPylation in these cell types which were then studied in cerebral organoids by overexpressing the FICD wt and E234G highly active mutant. This led to the accelerated differentiation of progenitors into the neurons. Overall our approach is suitable for identification of the AMPylated proteins in living cells and led to characterization of FICD function.

2024-05-21 | PXD008394 | Pride

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Project description:The AMPylation is novel protein post-translation modification. In consist of attachment of the adenosine monophosphate onto the serine, threonine or tyrosine amino acid residues of the protein. This process is catalysed by bacterial effectors or in human by protein FICD (also called HYPE) which contain conserved fic domain. In our study we have focused on identification of the unknown AMPylated proteins and their function in human cells (HeLa, A549, SH-SY5Y, iPSCs, NPCs and neurons) and cerebral organoids (COs). For this purpose we designed and synthesized the small molecule N6-propargyl phosphoramidate adenosine (pro-N6pA) which was used for in situ labelling of the AMPylation in cells and COs. Subsequnetly the the propargyl tag was employed for preparation of chemical-proteomic samples which were measured by LC-MS/MS. This approach confirmed the known AMPylation of heat shock protein HSPA5 and found diverse group of novel AMPylation targets. Several cathepsins found as AMPylation targets were further characterized by identification of AMP binding site. Based on our chemical proteomic data we found out that neurons and neuronal like cells contain distinct AMPylatino pattern from other studied cell types. This also suggested the importance of the AMPylation in these cell types which were then studied in cerebral organoids by overexpressing the FICD wt and E234G highly active mutant. This led to the accelerated differentiation of progenitors into the neurons. Overall our approach is suitable for identification of the AMPylated proteins in living cells and led to characterization of FICD function.

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HYPE mediated AMPylation profiling in human lysates

Project description:We describe here the first example of global chemoproteomic screening and substrate validation for HYPE mediated AMPylation in mammalian cell lysate. Through quantitative mass spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification we identified a total of 27 AMPylated proteins, including the previously validated substrate, ER chaperone BiP (HSPA5), along with novel substrates involved in pathways of gene expression, ATP biosynthesis and maintenance of cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

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2020-12-08 | PXD022078 | Pride

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Project description:AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology we here introduce a novel UMP pronucleotide probe and utilize it in the profiling of uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases which can largely differ compared to their in vitro activity.

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