Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties.

Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

Excessive tubulin glutamylation leads to progressive cone-rod dystrophy and loss of outer segment integrity.

Mutations in Cytosolic Carboxypeptidase-like Protein 5 (CCP5) are associated with vision loss in humans. To decipher the mechanisms behind CCP5-associated blindness, we generated a novel mouse model lacking CCP5. In this model, we found that increased tubulin glutamylation led to progressive cone-rod dystrophy, with cones showing a more pronounced and earlier functional loss than rod photoreceptors. The observed functional reduction was not due to cell death, levels, or the mislocalization of major phototransduction proteins. Instead, the increased tubulin glutamylation caused shortened photoreceptor axonemes and the formation of numerous abnormal membranous whorls that disrupted the integrity of photoreceptor outer segments (OS). Ultimately, excessive tubulin glutamylation led to the progressive loss of photoreceptors, affecting cones more severely than rods. Our results highlight the importance of maintaining tubulin glutamylation for normal photoreceptor function. Furthermore, we demonstrate that murine cone photoreceptors are more sensitive to disrupted tubulin glutamylation levels than rods, suggesting an essential role for axoneme in the structural integrity of the cone outer segment. This study provides valuable insights into the mechanisms of photoreceptor diseases linked to excessive tubulin glutamylation.

msqrob2PTM: Differential Abundance and Differential Usage Analysis of MS-Based Proteomics Data at the Posttranslational Modification and Peptidoform Level.

In the era of open-modification search engines, more posttranslational modifications than ever can be detected by LC-MS/MS-based proteomics. This development can switch proteomics research into a higher gear, as PTMs are key in many cellular pathways important in cell proliferation, migration, metastasis, and aging. However, despite these advances in modification identification, statistical methods for PTM-level quantification and differential analysis have yet to catch up. This absence can partly be explained by statistical challenges inherent to the data, such as the confounding of PTM intensities with its parent protein abundance. Therefore, we have developed msqrob2PTM, a new workflow in the msqrob2 universe capable of differential abundance analysis at the PTM and at the peptidoform level. The latter is important for validating PTMs found as significantly differential. Indeed, as our method can deal with multiple PTMs per peptidoform, there is a possibility that significant PTMs stem from one significant peptidoform carrying another PTM, hinting that it might be the other PTM driving the perceived differential abundance. Our workflows can flag both differential peptidoform abundance (DPA) and differential peptidoform usage (DPU). This enables a distinction between direct assessment of differential abundance of peptidoforms (DPA) and differences in the relative usage of peptidoforms corrected for corresponding protein abundances (DPU). For DPA, we directly model the log2-transformed peptidoform intensities, while for DPU, we correct for parent protein abundance by an intermediate normalization step which calculates the log2-ratio of the peptidoform intensities to their summarized parent protein intensities. We demonstrated the utility and performance of msqrob2PTM by applying it to datasets with known ground truth, as well as to biological PTM-rich datasets. Our results show that msqrob2PTM is on par with, or surpassing the performance of, the current state-of-the-art methods. Moreover, msqrob2PTM is currently unique in providing output at the peptidoform level.

USP36 inhibits apoptosis by deubiquitinating cIAP1 and survivin in colorectal cancer cells.

Chemotherapeutic agents for treating colorectal cancer (CRC) primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of CRC. Among the DUBs, ubiquitin-specific protease 36 (USP36) is upregulated in CRC. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11-linked ubiquitin chains from cIAP1 and lysine-48-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-second mitochondria-derived activator of caspase complex and promotes receptor-interacting protein kinase 1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in CRC progression and is a potential therapeutic target.

Essential role of CD155 glycosylation in functional binding to DNAM-1 on natural killer cells.

The cluster of differentiation 155 (CD155) is highly expressed on tumor cells and augments or inhibits the cytotoxic activities of natural killer (NK) cells and T cells through its receptor ligands DNAX accessory molecule 1 (DNAM-1) and T-cell immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), respectively. Although CD155 is heavily glycosylated, the role of glycosylation of CD155 in the cytotoxic activity of effector lymphocytes remains unknown. Here, we show that the N-linked glycosylation at residue 105 (N105 glycosylation) in the first Ig-like domain of CD155 is involved in the binding of CD155 to both DNAM-1 and TIGIT. The N105 glycosylation also plays an essential role to induce signaling in both DNAM-1 and TIGIT reporter cells. Moreover, we show that the N105 glycosylation of CD155 contributes preferentially to the DNAM-1-mediated activating signal over the TIGIT-mediated inhibitory signal in NK cells. Our results demonstrated the important role of the N105 glycosylation of CD155 in DNAM-1 and TIGIT functions and shed new light on the understanding of tumor immune responses.

Posttranslational modification and heme cavity architecture of human eosinophil peroxidase-insights from first crystal structure and biochemical characterization.

Eosinophil peroxidase (EPO) is the most abundant granule protein exocytosed by eosinophils, specialized human phagocytes. Released EPO catalyzes the formation of reactive oxidants from bromide, thiocyanate, and nitrite that kill tissue-invading parasites. However, EPO also plays a deleterious role in inflammatory diseases, making it a potential pharmacological target. A major hurdle is the high similarity to the homologous myeloperoxidase (MPO), which requires a detailed understanding of the small structural differences that can be used to increase the specificity of the inhibitors. Here, we present the first crystal structure of mature leukocyte EPO at 1.6 Å resolution together with analyses of its posttranslational modifications and biochemical properties. EPO has an exceptionally high number of positively charged surface patches but only two occupied glycosylation sites. The crystal structure further revealed the existence of a light (L) and heavy (H) chain as a result of proteolytic cleavage. Detailed comparison with the structure of human MPO allows us to identify differences that may contribute to the known divergent enzymatic properties. The crystal structure revealed fully established ester links between the prosthetic group and the protein, the comparably weak imidazolate character of the proximal histidine, and the conserved structure of the catalytic amino acids and Ca2+-binding site. Prediction of the structure of unprocessed proeosinophil peroxidase allows further structural analysis of the three protease cleavage sites and the potential pro-convertase recognition site in the propeptide. Finally, EPO biosynthesis and its biochemical and biophysical properties are discussed with respect to the available data from the well-studied MPO.

GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation.

GNE myopathy (GNEM) is a late-onset muscle atrophy, caused by mutations in the gene for the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). With an incidence of one to nine cases per million it is an ultra-rare, so far untreatable, autosomal recessive disease. Several attempts have been made to treat GNEM patients by oral supplementation with sialic acid precursors (e.g. N-acetylmannosamine, ManNAc) to restore sarcolemmal sialylation and muscle strength. In most studies, however, no significant improvement was observed. The lack of a suitable mouse model makes it difficult to understand the exact pathomechanism of GNEM and many years of research have failed to identify the role of GNE in skeletal muscle due to the lack of appropriate tools. We established a CRISPR/Cas9-mediated Gne-knockout cell line using murine C2C12 cells to gain insight into the actual role of the GNE enzyme and sialylation in a muscular context. The main aspect of this study was to evaluate the therapeutic potential of ManNAc and N-acetylneuraminic acid (Neu5Ac). Treatment of Gne-deficient C2C12 cells with Neu5Ac, but not with ManNAc, showed a restoration of the sialylation level back to wild type levels-albeit only with long-term treatment, which could explain the rather low therapeutic potential. We furthermore highlight the importance of sialic acids on myogenesis, for C2C12 Gne-knockout myoblasts lack the ability to differentiate into mature myotubes.

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Large-scale analysis of the N-terminal regulatory elements of the kinase domain in plant Receptor-like kinase family.

BACKGROUND: The N-terminal regulatory element (NRE) of Receptor-like kinases (RLKs), consisting of the juxtamembrane segment in receptor kinases (RKs) and the N-terminal extension segment in RLCKs, is a crucial component that regulates the activities of these proteins. However, the features and functions of the NRE have remained largely unexplored. Herein, we comprehensively analyze 510,233 NRE sequences in RLKs from 528 plant species, using information theory and data mining techniques to unravel their common characteristics and diversity. We also use recombinant RKs to investigate the function of the NRE in vitro. RESULTS: Our findings indicate that the majority of NRE segments are around 40-80 amino acids in length and feature a serine-rich region and a 14-amino-acid consensus sequence, 'FSYEELEKAT[D/N]NF[S/D]', which contains a characteristic α-helix and ST motif that connects to the core kinase domain. This conserved signature sequence is capable of suppressing FERONIA's kinase activity. A motif discovery algorithm identifies 29 motifs with highly conserved phosphorylation sites in RK and RLCK classes, especially the motif 'VGPWKpTGLpSGQLQKAFVTGVP' in LRR-VI-2 class. Phosphorylation of an NRE motif in an LRR-VI-2 member, MDIS1, modulates the auto-phosphorylation of its co-receptor, MIK1, indicating the potential role of NRE as a 'kinase switch' in RLK activation. Furthermore, the characterization of phosphorylatable NRE motifs improves the accuracy of predicting phosphorylatable sites. CONCLUSIONS: Our study provides a comprehensive dataset to investigate NRE segments from individual RLKs and enhances our understanding of the underlying mechanisms of RLK signal transduction and kinase activation processes in plant adaptation.

Unravelling HDAC Selectivity for Erasing Acetyl Mark on Lys-5 of Histone H2B.

The reversible acetylation of specific Lysine residues of histones plays crucial role in the epigenetic regulation of chromatin activity. Importantly, perturbations of acetylation-deacetylation dynamics have important implications for cancer and neurological disorders. There are 18 human HDACs including sirtuins. The site-selective acetyl eraser specificity of HDACs is poorly defined. Deciphering the site specificity preference of HDACs from a gamut of lysine in histones may be critical for targeted inhibitor development and delineation of regulatory mechanisms associated with chromatin. Here, we have interrogated the propensity of HDACs to erase acetyl mark at Lys-5 of H2B namely, H2BK5Ac engineered by a peptide ligation reaction catalyzed by transpeptidase sortase. HDACs and Sirtuins were individually over-expressed in HEK293 cells and the deacetylation propensity of respective cell lysates was evaluated against H2BK5Ac for initial screening of potential acetyl erasers. This screen indicated HDAC1 as the prime eraser of acetyl mark in H2BK5Ac. The propensity of HDAC1 to erase acetyl mark of H2BK5Ac was further probed using semisynthetic designer nucleosomes with whole cell lysates, recombinant enzyme, and specific inhibitors. Consistent with the above data, siRNA knockdown of HDAC1 and closely related HDAC3 in HEK293 cells prevented the loss of H2BK5 acetylation.

Synthesis and Semi-Synthesis of Alpha-Synuclein: Insight into the Chemical Complexity of Synucleinopathies.

The chemical rules governing protein folding have intrigued generations of researchers for decades. With the advent of artificial intelligence (AI), prediction of protein structure has improved tremendously. However, there is still a level of analysis that is only possible through wet laboratory experiments, especially in respect to the investigation of the pathological effect of mutations and posttranslational modifications (PTMs) on proteins of interest. This requires the availability of pure peptides and proteins in sufficient quantities for biophysical, biochemical, and functional studies. In this context, chemical protein synthesis and semi-synthesis are powerful tools in protein research, which help to enlighten the role of protein modification in the physiology and pathology of proteins. A protein of high interest in the field of biomedicine is alpha-synuclein (aSyn), a protein deeply associated with several devastating neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple systems atrophy (MSA). Here, we describe several methods and pathways to synthesize native or modified aSyn, and discuss how these approaches enable us to address pathological mechanisms that may open novel perspectives for therapeutic intervention.

The mechanism of RGS5 regulating gastric cancer mismatch repair protein.

The incidence and mortality rates of gastric cancer (GC) remain alarmingly high worldwide, imposing a substantial healthcare burden. In this study, we utilized data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A 4-gene prognostic model was developed to predict patient prognosis, and its accuracy was validated across multiple datasets. Patients with a low-risk score exhibited improved prognosis, elevated tumor mutation burden, heightened sensitivity to both immunotherapy and conventional chemotherapy. Notably, our investigation revealed that the key gene RGS5 positively modulates the expression of mismatch repair proteins via c-Myc. Furthermore, co-immunoprecipitation (COIP) assays demonstrated the interaction between RGS5 and c-Myc. Additionally, we confirmed that RGS5 regulates c-Myc through the ubiquitin-proteasome pathway. Moreover, RGS5 was identified as a positive regulator of PD-L1 expression and exhibited a negative correlation with the majority of immune cells. These findings underscore the potential of RGS5 as a novel biomarker and therapeutic target in the context of GC.

Spatial arrangement, polarity, and posttranslational modifications of the microtubule system in the Drosophila eye.

We have analyzed the organization of the microtubule system in photoreceptor cells and pigment cells within the adult Drosophila compound eye. Immunofluorescence localization of tubulin and of Short stop, a spectraplakin that has been reported to be involved in the anchorage of microtubule minus ends at the membrane, suggests the presence of non-centrosomal microtubule-organizing centers at the distal tip of the visual cells. Ultrastructural analyses confirm that microtubules emanate from membrane-associated plaques at the site of contact with cone cells and that all microtubules are aligned in distal-proximal direction within the photoreceptor cells. Determination of microtubule polarities demonstrated that about 95% of the microtubules in photoreceptor cells are oriented with their plus end in the direction of the synapse. Pigment cells in the eye contain only microtubules aligned in distal-proximal direction, with their plus end pointing towards the retinal floor. There, two populations of microtubules can be distinguished, single microtubules and bundled microtubules, the latter associated with actin filaments. Whereas microtubules in both photoreceptor cells and pigment cells are acetylated and mono/bi-glutamylated on α-tubulin, bundled microtubules in pigment cells are apparently also mono/bi-glutamylated on ß-tubulin, providing the possibility of binding different microtubule-associated proteins.

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling.

BACKGROUND: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems-the ubiquitin proteasome system (UPS) and macroautophagy-mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless, how Ate1 operates through this intricate mechanism remains elusive. METHODS: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofluorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform were used as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofluorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy. RESULTS: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. CONCLUSIONS: Our findings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.

Deubiquitinating enzymes: potential regulators of the tumor microenvironment and implications for immune evasion.

Ubiquitination and deubiquitination are important forms of posttranslational modification that govern protein homeostasis. Deubiquitinating enzymes (DUBs), a protein superfamily consisting of more than 100 members, deconjugate ubiquitin chains from client proteins to regulate cellular homeostasis. However, the dysregulation of DUBs is reportedly associated with several diseases, including cancer. The tumor microenvironment (TME) is a highly complex entity comprising diverse noncancerous cells (e.g., immune cells and stromal cells) and the extracellular matrix (ECM). Since TME heterogeneity is closely related to tumorigenesis and immune evasion, targeting TME components has recently been considered an attractive therapeutic strategy for restoring antitumor immunity. Emerging studies have revealed the involvement of DUBs in immune modulation within the TME, including the regulation of immune checkpoints and immunocyte infiltration and function, which renders DUBs promising for potent cancer immunotherapy. Nevertheless, the roles of DUBs in the crosstalk between tumors and their surrounding components have not been comprehensively reviewed. In this review, we discuss the involvement of DUBs in the dynamic interplay between tumors, immune cells, and stromal cells and illustrate how dysregulated DUBs facilitate immune evasion and promote tumor progression. We also summarize potential small molecules that target DUBs to alleviate immunosuppression and suppress tumorigenesis. Finally, we discuss the prospects and challenges regarding the targeting of DUBs in cancer immunotherapeutics and several urgent problems that warrant further investigation.

Transcription Factor TAL1 in Erythropoiesis.

Lineage-specific transcription factors (TFs) regulate differentiation of hematopoietic stem cells (HSCs). They are decisive for the establishment and maintenance of lineage-specific gene expression programs during hematopoiesis. For this they create a regulatory network between TFs, epigenetic cofactors, and microRNAs. They activate cell-type specific genes and repress competing gene expression programs. Disturbance of this process leads to impaired lineage fidelity and diseases of the blood system. The TF T-cell acute leukemia 1 (TAL1) is central for erythroid differentiation and contributes to the formation of distinct gene regulatory complexes in progenitor cells and erythroid cells. A TAL1/E47 heterodimer binds to DNA with the TFs GATA-binding factor 1 and 2 (GATA1/2), the cofactors LIM domain only 1 and 2 (LMO1/2), and LIM domain-binding protein 1 (LDB1) to form a core TAL1 complex. Furthermore, cell-type-dependent interactions of TAL1 with other TFs such as with runt-related transcription factor 1 (RUNX1) and Kruppel-like factor 1 (KLF1) are established. Moreover, TAL1 activity is regulated by the formation of TAL1 isoforms, posttranslational modifications (PTMs), and microRNAs. Here, we describe the function of TAL1 in normal hematopoiesis with a focus on erythropoiesis.

Posttranslational regulatory mechanism of PD-L1 in cancers and associated opportunities for novel small-molecule therapeutics.

Despite the tremendous progress in cancer research over the past few decades, effective therapeutic strategies are still urgently needed. Accumulating evidence suggests that immune checkpoints are the cause of tumor immune escape. PD-1/PD-L1 are among them. Posttranslational modification is the most critical step for protein function, and the regulation of PD-L1 by small molecules through posttranslational modification is highly valuable. In this review, we discuss the mechanisms of tumor cell immune escape and several posttranslational modifications associated with PD-L1 and describe examples in which small molecules can regulate PD-L1 through posttranslational modifications. Herein, we propose that the use of small molecule compounds that act by inhibiting PD-L1 through posttranslational modifications is a promising therapeutic approach with the potential to improve clinical outcomes for cancer patients.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.