Lysine 2-hydroxyisobutyrylation (K hib) is a novel posttranslational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of K hib in Proteus mirabilis (P. mirabilis). By combining affinity enrichment with two-dimensional liquid chromatography and high-resolution mass spectrometry, 4735 2-hydroxyisobutyrylation sites were identified on 1051 proteins in P. mirabilis. These proteins bearing modifications were further characterized in abundance, distribution and functions. The interaction networks and domain architectures of these proteins with high confidence were revealed using bioinformatic tools. Our data demonstrate that many 2-hydroxyisobutyrylated proteins are involved in metabolic pathways, such as purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. The extensive distribution of K hib also indicates that the modification may play important influence to bacterial metabolism. The speculation is further supported by the observation that carbon sources can influence the occurrence of K hib. Furthermore, we demonstrate that 2-hy-droxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that K hib may change the binding formation of ENO and its substrate 2-phospho-D-glycerate (2PG) and cause the substrate far from the active sites of enzyme. We hope this first comprehensive analysis of nonhistone K hib in prokaryotes is valuable for further functional investigation of this modification.

Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.

Background Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post‐translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. Methods Immunoprecipitation and glutathione‐S‐transferase pull‐down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP‐associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF‐mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live‐cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. Results Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti‐AcK471‐Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin‐K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin‐K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin‐K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471‐acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin‐bundling activity. Clinically, high levels of AcK471‐Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease‐free survival of ESCC patients. Conclusions Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin‐K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin‐bundling activity.