Function and regulation of RGS family members in solid tumours: a comprehensive review.

G protein-coupled receptors (GPCRs) play a key role in regulating the homeostasis of the internal environment and are closely associated with tumour progression as major mediators of cellular signalling. As a diverse and multifunctional group of proteins, the G protein signalling regulator (RGS) family was proven to be involved in the cellular transduction of GPCRs. Growing evidence has revealed dysregulation of RGS proteins as a common phenomenon and highlighted the key roles of these proteins in human cancers. Furthermore, their differential expression may be a potential biomarker for tumour diagnosis, treatment and prognosis. Most importantly, there are few systematic reviews on the functional/mechanistic characteristics and clinical application of RGS family members at present. In this review, we focus on the G-protein signalling regulator (RGS) family, which includes more than 20 family members. We analysed the classification, basic structure, and major functions of the RGS family members. Moreover, we summarize the expression changes of each RGS family member in various human cancers and their important roles in regulating cancer cell proliferation, stem cell maintenance, tumorigenesis and cancer metastasis. On this basis, we outline the molecular signalling pathways in which some RGS family members are involved in tumour progression. Finally, their potential application in the precise diagnosis, prognosis and treatment of different types of cancers and the main possible problems for clinical application at present are discussed. Our review provides a comprehensive understanding of the role and potential mechanisms of RGS in regulating tumour progression. Video Abstract.

CHDH, a key mitochondrial enzyme, plays a diagnostic role in metabolic disorders diseases and tumor progression.

Human choline dehydrogenase (CHDH) is a transmembrane protein located in mitochondria. CHDH has been shown to be one of the important catalytic enzymes that catalyze the oxidation of choline to betaine and is involved in mitochondrial autophagy after mitochondrial damage. In recent years, an increasing number of studies have focused on CHDH and found a close association with the pathogenesis of various diseases, including tumor prognosis. Here we summarized the genomic localization, protein structure and basic functions of CHDH and discuss the progress of CHDH research in metabolic disorders and other diseases. Moreover, we described the regulatory role of CHDH on the progression of different types of malignant tumors. In addition, major pathogenic mechanisms of CHDH in multiple diseases may be associated with single nucleotide polymorphism (SNP). We look forward to providing new strategies and basis for clinical diagnosis and prognosis prediction of diseases by diagnosing SNP loci of CHDH genes. Our work evaluates the feasibility of CHDH as a molecular marker relevant to the diagnosis of some metabolic disorders diseases and tumors, which may provide new targets for the treatment of related diseases and tumors.

Integrated multi-omics analyses reveal Jorunnamycin A as a novel suppressor for muscle-invasive bladder cancer by targeting FASN and TOP1.

BACKGROUND: Bladder cancer is a urological carcinoma with high incidence, among which muscle invasive bladder cancer (MIBC) is a malignant carcinoma with high mortality. There is an urgent need to develop new drugs with low toxicity and high efficiency for MIBC because existing medication has defects, such as high toxicity, poor efficacy, and side effects. Jorunnamycin A (JorA), a natural marine compound, has been found to have a high efficiency anticancer effect, but its anticancer function and mechanism on bladder cancer have not been studied. METHODS: To examine the anticancer effect of JorA on MIBC, Cell Counting Kit 8, EdU staining, and colony formation analyses were performed. Moreover, a xenograft mouse model was used to verify the anticancer effect in vivo. To investigate the pharmacological mechanism of JorA, high-throughput quantitative proteomics, transcriptomics, RT-qPCR, western blotting, immunofluorescence staining, flow cytometry, pulldown assays, and molecular docking were performed. RESULTS: JorA inhibited the proliferation of MIBC cells, and the IC50 of T24 and UM-UC-3 was 0.054 and 0.084 µM, respectively. JorA-induced significantly changed proteins were enriched in "cancer-related pathways" and "EGFR-related signaling pathways", which mainly manifested by inhibiting cell proliferation and promoting cell apoptosis. Specifically, JorA dampened the DNA synthesis rate, induced phosphatidylserine eversion, and inhibited cell migration. Furthermore, it was discovered that fatty acid synthase (FASN) and topoisomerase 1 (TOP1) are the JorA interaction proteins. Using DockThor software, the 3D docking structures of JorA binding to FASN and TOP1 were obtained (the binding affinities were - 8.153 and - 7.264 kcal/mol, respectively). CONCLUSIONS: The marine compound JorA was discovered to have a specific inhibitory effect on MIBC, and its potential pharmacological mechanism was revealed for the first time. This discovery makes an important contribution to the development of new high efficiency and low toxicity drugs for bladder cancer therapy.

Homeobox A3 and KDM6A cooperate in transcriptional control of aerobic glycolysis and glioblastoma progression.

BACKGROUND: Alterations in transcriptional regulators of glycolytic metabolism have been implicated in brain tumor growth, but the underlying molecular mechanisms remain poorly understood. METHODS: Knockdown and overexpression cells were used to explore the functional roles of HOXA3 in cell proliferation, tumor formation, and aerobic glycolysis. Chromatin immunoprecipitation, luciferase assays, and western blotting were performed to verify the regulation of HK2 and PKM2 by HOXA3. PLA, Immunoprecipitation, and GST-pull-down assays were used to examine the interaction of HOXA3 and KDM6A. RESULTS: We report that transcription factor homeobox A3 (HOXA3), which is aberrantly highly expressed in glioblastoma (GBM) patients and predicts poor prognosis, transcriptionally activates aerobic glycolysis, leading to a significant acceleration in cell proliferation and tumor growth. Mechanically, we identified KDM6A, a lysine-specific demethylase, as an important cooperator of HOXA3 in regulating aerobic glycolysis. HOXA3 activates KDM6A transcription and recruits KDM6A to genomic binding sites of glycolytic genes, targeting glycolytic genes for transcriptional activation by removing the suppressive histone modification H3K27 trimethylation. Further evidence demonstrates that HOXA3 requires KDM6A for transcriptional activation of aerobic glycolysis and brain tumor growth. CONCLUSIONS: Our findings provide a novel molecular mechanism linking HOXA3-mediated transactivation and KDM6A-coupled H3K27 demethylation in regulating glucose metabolism and GBM progression.

Mitoxantrone triggers immunogenic prostate cancer cell death via p53-dependent PERK expression.

BACKGROUND: Mitoxantrone (MTX) is a synthetic compound used as a second line chemotherapeutic drug for prostate cancer. It has been reported to trigger immunogenic cell death (ICD) in animal model studies, but the underlying mechanism is not fully understood yet, especially not in prostate cancer cells. METHODS: ICD was determined by assessing the release of damage-associated molecular patterns (DAMPs) in the prostate cancer-derived cell lines LNCaP, 22RV1 and PC-3. Short hairpin RNAs (shRNAs) were used to knock down target gene expression. Phagocytosis was assessed using a dual labeling technology in dendric cells co-cultured with cancer cells. The PERK gene promoter was cloned for dual luciferase assays. Chromatin immunoprecipitation (ChIP) was used to determine p53 protein-DNA binding activity. Immunocompetent mice and murine RM-1 prostate cancer cells were used for vaccination experiments. RESULTS: MTX treatment induced typical characteristics of DAMP release, including increased cell surface exposure of calreticulin (CALR), and extracellular release of ATP and high mobility group box-1 (HMGB1) protein. MTX also enhanced phagocytosis by dendritic cells. Moreover, MTX treatment increased eukaryotic initiation factor 2α (eIF2α) S51 phosphorylation, which was reduced when PERK and GCN2 were silenced using shRNAs. In addition, PERK or GCN2 silencing significantly reduced MTX-induced release of DAMPs in vitro and anti-tumor immunity in vivo. MTX treatment also resulted in dendritic cell activation in mice, which was attenuated when PERK or GCN2 were silenced in cancer cells used for vaccination. Further analysis revealed that PERK and GCN2 expression was enhanced by MTX treatment, of which PERK, but not GCN2, was enhanced via a p53-dependent mechanism. CONCLUSION: MTX triggers ICD by activating eIF2α via PERK/GCN2 upregulation in prostate cancer cells. MTX-induced PERK expression upregulation depends on the p53 pathway, while that of GCN2 requires further investigation.

Crystal structure of sulfonic peroxiredoxin Ahp1 in complex with thioredoxin Trx2 mimics a conformational intermediate during the catalytic cycle.

Peroxiredoxin (Prx) is a thiol-based peroxidase that eliminates reactive oxygen species to avoid oxidative damage. Alkyl hydroperoxide reductase Ahp1 is a novel and specific typical 2-cysteine Prx. Here, we present the crystal structure of sulfonic Ahp1 complexed with thioredoxin Trx2 at 2.12 Å resolution. This structure implies that the transient Ahp1-Trx2 complex during the catalytic cycle already have an ability to decompose the peroxides. Structural analysis reveals that the segment glutamine23-lysine32 juxtaposed to the resolving cysteine (CR) of Ahp1 moves inward to generate a compact structure upon peroxidatic cysteine (CP) overoxidation, resulting in the breakdown of several conserved hydrogen bonds formed by Ahp1-Trx2 complex interaction. Structural comparisons suggest that the structure of sulfonic Ahp1 represents a novel conformation of Ahp1, which can mimic a conformational intermediate between the reduced and oxidized forms. Therefore, this study may provide a new structural insight into the intermediate state in which the segment glutamine23-lysine32 juxtaposed to the cysteine31 (CR) undergoes a conformational change upon cysteine62 (CP) oxidation to prepare for the formation of an intermolecular CP-CR disulfide bond during Ahp1 catalytic cycle.

New structural insights into the recognition of undamaged splayed-arm DNA with a single pair of non-complementary nucleotides by human nucleotide excision repair protein XPA.

XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.

Structural characterization of the redefined DNA-binding domain of human XPA.

XPA (xeroderma pigmentosum complementation group A), a key scaffold protein in nucleotide excision repair (NER) pathway, is important in DNA damage verification and repair proteins recruitment. Earlier studies had mapped the minimal DNA-binding domain (MBD) of XPA to a region corresponding to residues 98-219. However, recent studies indicated that the region involving residues 98-239 is the redefined DNA-binding domain (DBD), which binds to DNA substrates with a much higher binding affinity than MBD and possesses a nearly identical binding affinity to the full-length XPA protein. However, the structure of the redefined DBD domain of XPA (XPA-DBD) remains to be investigated. Here, we present the crystal structure of XPA-DBD at 2.06 Šresolution. Structure of the C-terminal region of XPA has been extended by 21 residues (Arg211-Arg231) as compared with previously reported MBD structures. The structure reveals that the C-terminal extension (Arg211-Arg231) is folded as an α-helix with multiple basic residues. The positively charged surface formed in the last C-terminal helix suggests its critical role in DNA binding. Further structural analysis demonstrates that the last C-terminal region (Asp217-Thr239) of XPA-DBD might undergo a conformational change to directly bind to the DNA substrates. This study provides a structural basis for understanding the possible mechanism of enhanced DNA-binding affinity of XPA-DBD.

Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation.

UHRF1 plays multiple roles in regulating DNMT1-mediated DNA methylation maintenance during DNA replication. The UHRF1 C-terminal RING finger functions as an ubiquitin E3 ligase to establish histone H3 ubiquitination at Lys18 and/or Lys23, which is subsequently recognized by DNMT1 to promote its localization onto replication foci. Here, we present the crystal structure of DNMT1 RFTS domain in complex with ubiquitin and highlight a unique ubiquitin binding mode for the RFTS domain. We provide evidence that UHRF1 N-terminal ubiquitin-like domain (UBL) also binds directly to DNMT1. Despite sharing a high degree of structural similarity, UHRF1 UBL and ubiquitin bind to DNMT1 in a very distinct fashion and exert different impacts on DNMT1 enzymatic activity. We further show that the UHRF1 UBL-mediated interaction between UHRF1 and DNMT1, and the binding of DNMT1 to ubiquitinated histone H3 that is catalyzed by UHRF1 RING domain are critical for the proper subnuclear localization of DNMT1 and maintenance of DNA methylation. Collectively, our study adds another layer of complexity to the regulatory mechanism of DNMT1 activation by UHRF1 and supports that individual domains of UHRF1 participate and act in concert to maintain DNA methylation patterns.

Crystal structure and SUMO binding of Slx1-Slx4 complex.

The SLX1-SLX4 complex is a structure-specific endonuclease that cleaves branched DNA structures and plays significant roles in DNA recombination and repair in eukaryotic cells. The heterodimeric interaction between SLX1 and SLX4 is essential for the endonuclease activity of SLX1. Here, we present the crystal structure of Slx1 C-terminal zinc finger domain in complex with the C-terminal helix-turn-helix domain of Slx4 from Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals a conserved binding mechanism underling the Slx1-Slx4 interaction. Structural and sequence analyses indicate Slx1 C-terminal domain is actually an atypical C4HC3-type RING finger which normally possesses E3 ubiquitin ligase activity, but here is absolutely required for Slx1 interaction with Slx4. Furthermore, we found the C-terminal tail of S. pombe Slx1 contains a SUMO-interacting motif and can recognize Pmt3 (S. pombe SUMO), suggesting that Slx1-Slx4 complex could be recruited by SUMOylated protein targets to take part in replication associated DNA repair processes.

The N-terminal ß-sheet of peroxiredoxin 4 in the large yellow croaker Pseudosciaena crocea is involved in its biological functions.

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that exhibit peroxidase and peroxynitrite reductase activities involved in the reduction of reactive oxygen species. The peroxiredoxin Prx4 from the large yellow croaker Pseudosciaena crocea is a typical 2-Cys Prx with an N-terminal signal peptide. We solved the crystal structure of Prx4 at 1.90 Å and revealed an N-terminal antiparallel ß-sheet that contributes to the dimer interface. Deletion of this ß-sheet decreased the in vitro peroxidase activity to about 50% of the wild-type. In vivo assays further demonstrated that removal of this ß-sheet led to some impairment in the ability of Prx4 to negatively regulate nuclear factor-κB (NF-κB) activity and to perform its role in anti-bacterial immunity. These results provide new insights into the structure and function relationship of a peroxiredoxin from bony fish.

Structural snapshots of yeast alkyl hydroperoxide reductase Ahp1 peroxiredoxin reveal a novel two-cysteine mechanism of electron transfer to eliminate reactive oxygen species.

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C(P)) to Cys-62 and the resolving cysteine (C(R)) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Šresolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C(P) and C(R). An intermolecular C(P)-C(R) disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.

Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33.

The yeast Saccharomyces cerevisiae mitochondrial matrix factor Mmf1, a member in the YER057c/Yigf/Uk114 family, participates in isoleucine biosynthesis and mitochondria maintenance. Mmf1 physically interacts with another mitochondrial matrix protein Mam33, which is involved in the sorting of cytochrome b2 to the intermembrane space as well as mitochondrial ribosomal protein synthesis. To elucidate the structural basis for their interaction, we determined the crystal structures of Mmf1 and Mam33 at 1.74 and 2.10 Å, respectively. Both Mmf1 and Mam33 adopt a trimeric structure: each subunit of Mmf1 displays a chorismate mutase fold with a six-stranded ß-sheet flanked by two α-helices on one side, whereas a subunit of Mam33 consists of a twisted six-stranded ß-sheet surrounded by five α-helices. Biochemical assays combined with structure-based computational simulation enable us to model a putative complex of Mmf1-Mam33, which consists of one Mam33 trimer and two tandem Mmf1 trimers in a head-to-tail manner. The two interfaces between the ring-like trimers are mainly composed of electrostatic interactions mediated by complementary negatively and positively charged patches. These results provided the structural insights into the putative function of Mmf1 during mitochondrial protein synthesis via Mam33, a protein binding to mitochondrial ribosomal proteins.

Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103.

BACKGROUND: The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: alpha-, beta-, gamma-, delta-, epsilon-, zeta- CAs, with beta-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of beta-CA Nce103/YNL036W. RESULTS: We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 A resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable. CONCLUSION: The quaternary structure of Nce103 resembles the typical plant type beta-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.