GG Motif Research Articles

Targeting m7G-enriched circKDM1A prevents colorectal cancer progression.

Plenty of circRNAs have been reported to play an important role in colorectal cancer (CRC), while the reason of abnormal circRNA expression in cancer still keep elusive. Here, we found that m7G RNA modifications were enriched in some circRNAs, these m7G modifications in circRNAs were catalyzed by METTL1, and the GG motif was the main site preference for m7G modifications in circRNAs. We further confirmed that METTL1 played a cancer-promoting role in CRC. We then screened a highly expressed circRNA, called circKDM1A, and found that METTL1 prevented the degradation of circKDM1A by m7G modification. CircKDM1A was further verified to promote proliferation, invasion and migration of CRC in vivo and in vitro. Its cancer-promoting ability was weakened after the m7G site mutation. CircKDM1A was verified to activate AKT pathway by upregulating PDK1, consequently promoting CRC progression. These results suggest that m7G-modified circRNA promotes CRC progression via activating AKT pathway. Our study uncovers an essential physiological function and mechanism of METTL1-mediated m7G modification in the regulation of circRNA stability and cancer progression.

Generation of antibody-drug conjugates by proximity-driven acyl transfer and sortase-mediated ligation.

We report a sortase-based site-specific antibody-drug conjugation strategy, which involves an affinity peptide-directed acyl transfer reaction and sortase-mediated peptide ligation. Through the affinity peptide-mediated acyl transfer reaction, an LPXTG-containing peptide is conjugated to a specific Lys side chain of an antibody. Under the assistance of sortase, a protein drug bearing a GG motif reacts specifically with the LPXTG moiety to produce an antibody-drug conjugate. Our strategy for antibody conjugation can be applied not only to chemically synthesized drugs, but also to biologically expressed proteins, and will provide a new sortase-based strategy for the preparation of antibody-drug conjugates.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

BackgroundWith the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5′ GG motif for efficient initiation. The resulting transcripts bear a 5′ GG motif, which significantly constrains the number of targetable sites in the silkworm genome.ResultsIn this study, we used the T7 promoter to add two supernumerary G residues to the 5′ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5′ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5′ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5′ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5′ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5′ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation.ConclusionsIn summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.

CRISPR technologies have overwhelmingly relied on the Streptococcus pyogenes Cas9 (SpyCas9), with its consensus NGG and less preferred NAG and NGA protospacer-adjacent motifs (PAMs). Here, we report that SpyCas9 also recognizes sequences within an N(A/C/T)GG motif. These sequences were identified on the basis of preferential enrichment in a growth-based screen in Escherichia coli. DNA binding, cleavage, and editing assays in bacteria and human cells validated recognition, with activities paralleling those for NAG(A/C/T) PAMs and dependent on the first two PAM positions. Molecular-dynamics simulations and plasmid-clearance assays with mismatch-intolerant variants supported induced-fit recognition of an extended PAM by SpyCas9 rather than recognition of NGG with a bulged R-loop. Last, the editing location for SpyCas9-derived base editors could be shifted by one nucleotide by selecting between (C/T)GG and adjacent N(C/T)GG PAMs. SpyCas9 and its enhanced variants thus recognize a larger repertoire of PAMs, with implications for precise editing, off-target predictions, and CRISPR-based immunity.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

SummaryThe family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

Structural and functional characterization of Tomato SUMO1 gene.

Small ubiquitin-related modifier (SUMO) genes regulate various functions of target proteins through post-translational modification. The SUMO proteins have a similar 3-dimensional structure as that of ubiquitin proteins and occur through a cascade of enzymatic reactions. In the present study we have cloned a new SUMO gene from Tomato (Solanum lycopersicum L.), cv Saudi-1, named SlS-SUMO1 gene by PCR using specific primers. This gene has SUMO member's features such as C-terminal diglycine (GG) motif as processing site by ULP (ubiquitin-like SUMO protease) and has SUMO consensus ΨKXE/D sequence. Phylogenetic analysis showed that SlS-SUMO1 gene is highly conserved and homologous to Potatoes Ca-SUMO1 and Ca-SUMO2 genes based on sequence similarity. Expression protein of SlS-SUMO1 gene found to be localized in the nucleus, cytoplasm, and nuclear envelop or nuclear pore complex. SUMO conjugating enzyme SCE1a with SlS-SUMO1 protein co-expressed and co-localized in nucleus and formed nuclear subdomains. This study reported that the SlS-SUMO1 gene is a member of SUMO family and its SUMO protein processing using GG motif and activate and transport to nucleus through Sumoylation system in the plant cell.

Mapping of SUMOylated Proteins in Renal Cortical Collecting Duct (CCD) and Distal Convoluted Tubule (DCT) Cells

Kidney epithelial cells are continuously subjected to various forms of cellular stress, including osmotic, oxidative and mechanical stress. To respond and survive during stress, renal cells adapt using various defence mechanisms. One of these mechanisms is proposed to be protein post‐translational modification with the small‐ubiquitin‐related‐modifier (SUMO), which modulates protein and cellular function to counterbalance the subjected stress. In mammals, five SUMO isoforms (SUMO1‐5) are expressed, but their role in kidney function has never been addressed. The aim of this study was to profile SUMO1 and SUMO2 modified proteins in cortical collecting duct (mpkCCD) and distal convoluted tubule (mpkDCT) cells as a precursor to targeted approaches to assess SUMO function. A modified form of SUMO1 or SUMO2, with a histidine tag and a T‐K mutation preceding the carboxyl‐terminal GG motif (position where SUMO attaches to substrates) was expressed in mpkCCD or mpkDCT cells. SUMO levels in cells following heat shock, treatment with deSUMOylation inhibitors, or aldosterone were assessed. Following cell lysis under denaturing conditions, SUMO‐conjugated proteins were purified using the histidine tag, and after cleavage with the endoproteinase Lys‐C, a GG‐specific antibody was used to enrich SUMOylated peptides. Peptides were identified using LC‐MS/MS and analysed using Proteome Discoverer and MaxQuant. 1428 SUMO1 and 1957 SUMO2 modified sites were identified in mpkCCD cells, corresponding to 741 SUMO1 and 971 SUMO2 proteins, respectively. In mpkDCT cells, 248 SUMO1 and 258 SUMO2 modified sites corresponding to 173 SUMO1 and 174 SUMO2 proteins, respectively, were identified. 588 proteins for mpkCCD and 74 proteins for mpkDCT were SUMOylated by both SUMO1 and SUMO2 at the same site. A well established SUMOylation KxE motif and reversed KxD/E‐motif were identified. Although several classes of SUMOylated proteins were highlighted by gene ontology, the majority of SUMOylated proteins in mpkCCD/DCT cells were related to transcription. Furthermore, we determined that the levels of SUMOylation at a specific site on the proton and oligopeptide/antibiotic cotransporter protein, PEPT2, were greatly increased after aldosterone stimulation, suggesting that SUMOylation of proteins in the CCD or DCT is under physiological regulation. In conclusion, we assessed proteome‐wide SUMOylation in renal epithelial cells for the first time. These studies lay the foundation for ongoing work assessing the role of individual SUMOylation sites in protein and kidney function.Support or Funding InformationFunding is provided by the Novo Nordisk Foundation, the Lundbeck Foundation, the Danish Medical Research Council and the Foundation Leducq (to R.A.F). Q.W. is supported by the European Union Horizon 2020 Marie Skłodowska‐Curie Individual Fellowship (Project No. 705682) and the Danish Medical Research Council (Ref. No. 6110‐00118B).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A microcin processing peptidase-like protein of the cyanobacterium Synechococcus elongatus is essential for secretion of biofilm-promoting proteins.

Small secreted compounds, e.g. microcins, are characterized by a double-glycine (GG) secretion motif that is cleaved off upon maturation. Genomic analysis suggests that small proteins that possess a GG motif are widespread in cyanobacteria; however, the roles of these proteins are largely unknown. Using a biofilm-proficient mutant of the cyanobacterium Synechococcus elongatus PCC 7942 in which the constitutive biofilm self-suppression mechanism is inactivated, we previously demonstrated that four small proteins, Enable biofilm formation with a GG motif (EbfG1-4), each with a GG motif, enable biofilm formation. Furthermore, a peptidase belonging to the C39 family, Peptidase transporter enabling Biofilm (PteB), is required for secretion of these proteins. Here, we show that the microcin processing peptidase-like protein encoded by gene Synpcc7942_1127 is also required for biofilm development - inactivation of this gene in the biofilm-proficient mutant abrogates biofilm development. Additionally, this peptidase-like protein (denoted EbfE - enables biofilm formation peptidase) is required for secretion of the EbfG biofilm-promoting small proteins. Given their protein-domain characteristics, we suggest that PteB and EbfE take part in a maturation-secretion system, with PteB being located to the cell membrane while EbfE is directed to the periplasmic space via its secretion signal.

Structural study reveals the temperature-dependent conformational flexibility of Tk-PTP, a protein tyrosine phosphatase from Thermococcus kodakaraensis KOD1.

Protein tyrosine phosphatases (PTPs) originating from eukaryotes or bacteria have been under intensive structural and biochemical investigation, whereas archaeal PTP proteins have not been investigated extensively; therefore, they are poorly understood. Here, we present the crystal structures of Tk-PTP derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, in both the active and inactive forms. Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and α4−α5 loop regions, switching between the inactive and active forms. Through comprehensive analyses of Tk-PTP, including additional structural determination of the G95A mutant form, enzymatic activity assays, and structural comparison with the other archaeal PTP, it was revealed that the presence of the GG motif in the P-loop is necessary but not sufficient for the structural flexibility of Tk-PTP. It was also proven that Tk-PTP contains dual general acid/base residues unlike most of the other DUSP proteins, and that both the residues are critical in its phosphatase activity. This work provides the basis for expanding our understanding of the previously uncharacterized PTP proteins from archaea, the third domain of living organisms.

Structural Insights into the Glycine Pair Motifs in Type III Collagen.

Human type III collagen has been suggested to play vital roles in a series of pathophysiological conditions. Sequence analysis among major fibril-forming collagens (types I, II, and III) revealed that Gly-Gly pairs are a distinct sequence feature in type III collagen. This motif occurs more than five times as often in type III compared to type I and II collagens. We used an integrated computational modeling and biophysical approach to analyze the glycine pair motifs to understand how they govern the structure of type III collagen at the molecular level. Triple helical peptides to model the regions of type III collagen containing GG motifs were used to analyze structural and thermodynamic effects of GG incorporation into the collagen sequence. We found that when amino acids adjacent to a GG motif are charged, the collagen adopts a more flexible, random conformation. The GG motif led to altered hydrogen bond patterns and decreased global melting temperatures of the triple helical peptides. The local entropic destabilization effect of the glycine pair helps explain the difference in the flexibility between types I and III collagen fibrils. This finding reveals potential physiological roles of type III collagen in regulating the mechanical properties of collagen fibrils and may enable the design of future collagen-like materials with tunable mechanical properties.

Identification of high-efficiency 3'GG gRNA motifs in indexed FASTA files with ngg2.

CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3'GG motif, which substantially increases the efficiency of editing at all sites tested in C. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a python command-line tool, ngg2, to identify 3'GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes: Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3'GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3'GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3'GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3'GG editing sites in any species with an available genome sequence.

C-Terminal glycine-gated radical initiation by GTP 3',8-cyclase in the molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is an essential redox cofactor found in all kingdoms of life. Genetic mutations in the human Moco biosynthetic enzymes lead to a fatal metabolic disorder, Moco deficiency (MoCD). Greater than 50% of all human MoCD patients have mutations in MOCS1A, a radical S-adenosyl-l-methionine (SAM) enzyme involved in the conversion of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate. In MOCS1A, one of the frequently affected locations is the GG motif constituted of two consecutive Gly at the C-terminus. The GG motif is conserved among all MOCS1A homologues, but its role in catalysis or the mechanism by which its mutation causes MoCD was unknown. Here, we report the functional characterization of the GG motif using MoaA, a bacterial homologue of MOCS1A, as a model. Our study elucidated that the GG motif is essential for the activity of MoaA to produce 3',8-cH2GTP from GTP (GTP 3',8-cyclase), and that synthetic peptides corresponding to the C-terminal region of wt-MoaA rescue the GTP 3',8-cyclase activity of the GG-motif mutants. Further biochemical characterization suggested that the C-terminal tail containing the GG motif interacts with the SAM-binding pocket of MoaA, and is essential for the binding of SAM and subsequent radical initiation. In sum, these observations suggest that the C-terminal tail of MoaA provides an essential mechanism to trigger the free radical reaction, impairment of which results in the complete loss of catalytic function of the enzyme, and causes MoCD.

Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.

Flexible connection of the N‐terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency

ClpB reactivates aggregated proteins in cooperation with DnaK/J. The ClpB monomer contains two nucleotide-binding domains (D1, D2), a coiled-coil domain, and an N-terminal domain attached to D1 with a 17-residue-long unstructured linker containing a Gly-Gly motif. The ClpB-mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric ClpB, but the events preceding the translocation are poorly understood. The N-terminal domains form a ring surrounding the entrance to the channel and contribute to the aggregate binding. It was suggested that the N-terminal domain's mobility that is maintained by the unstructured linker might control the efficiency of aggregate reactivation. We produced seven variants of ClpB with modified sequence of the N-terminal linker. To increase the linker's conformational flexibility, we inserted up to four Gly next to the GG motif. To decrease the linker's flexibility, we deleted the GG motif and converted it into GP and PP. We found that none of the linker modifications inhibited the basal ClpB ATPase activity or its capability to form oligomers. However, the modified linker ClpB variants showed lower reactivation rates for aggregated glucose-6-phosphate dehydrogenase and firefly luciferase and a lower aggregate-binding efficiency than wt ClpB. We conclude that the linker does not merely connect the N-terminal domain, but it supports the chaperone activity of ClpB by contributing to the efficiency of aggregate binding and disaggregation. Moreover, our results suggest that selective pressure on the linker sequence may be crucial for maintaining the optimal efficiency of aggregate reactivation by ClpB.

The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1-C CRISPR2 is present in few strains but type 2-A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre-exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer-associated motif-shuffling demonstrated that the GG motif is sufficient to discriminate self and non-self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II-A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population.

Characterization of the C-Terminal Diglycine Motif of SUMO-1/3

A hallmark of small ubiquitin-related modifier (SUMO) is the production of a C-terminal tail containing diglycines (GGs), which are believed to be required for SUMOylation. Whether GGs are required components in SUMOylation remains unanswered experimentally. In this study we found that the SUMO-1/3-AA/-GS/-GN/-GA mutant can form sodium dodecyl sulfate (SDS)-dithiothreitol (DTT)-resistant complexes with cellular proteins, indicating that the GG motif is not strictly required for SUMOylation.

Studying the interactions of a platinum(ii) 9-aminoacridine complex with proteins and oligonucleotides by ESI-TOF MS

The interaction of a novel Pt complex, [Pt(dmba)(N9-9AA)(PPh(3))](+)1 (dmba = N,N-dimethylbenzylamine-κN,κC; 9AA = 9-aminoacridine), which exhibits anti-tumor activity, with certain key proteins has been monitored by ESI-MS. Also, the interaction of 1 with a designed double-stranded oligonucleotide containing the GG motif has been followed by mass spectrometry as well as by fluorimetry. The results obtained show the low interaction of 1 with the considered proteins and the absence of covalent interaction with the oligonucleotides, but the fluorimetric data confirm the π-π interaction of 1 with the double-stranded DNA, which is probably the reason of the previously reported activity of 1 in several tumor cell lines.

Crystallization and preliminary X-ray crystallographic studies of an oligomeric species of a refolded C39 peptidase-like domain of theEscherichia coliABC transporter haemolysin B

The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.

The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties

An EST, encoding a strawberry phytocystatin (PhyCys) obtained from a developing fruit of Fragariaxananassa cv. Elsanta has been characterized. The corresponding gene (Cyf1) had three introns interrupting its ORF that codes for a protein (FaCPI-1) of 235 amino acid residues with a putative signal peptide of 29 residues and an estimated molecular mass for the mature protein of 23.1 kDa. This protein contains, besides a C-terminal extension, several motifs conserved in all members of the PhyCys superfamily: (i) a GG and LARFAV-like motifs towards the N-terminal part of the protein; (ii) the reactive site QVVAG, and (iii) a conserved PW, downstream of the reactive site. Northern blot and in situ hybridization analyses indicated that the Cyf1 gene was expressed in fully expanded leaves, in roots and in achenes, but not in the receptacle (pseudocarp) during fruit development. The recombinant FaCPI-1 protein expressed in E. coli efficiently inhibited papain (K(i) 1.9 x 10(-9) M) and less so cathepsin H (K(i) 4.7 x 10(-7) M) and cathepsin B (K(i) 3.3 x 10(-6) M), and was a good inhibitor of the in vitro growth of phytopathogenic fungi Botrytis cinerea (EC(50): 1.90 microM) and Fusarium oxysporum (EC(50): 2.28 microM).

Functional analysis of diploid wheat rRNA promoter by transient expression

The rRNA gene promoter regions of three diploid wheats Triticum boeoticum Boiss, Triticum urartu Tum. ex Gandil, and Triticum monococcum L. have been sequenced and analyzed. It has been found that the rRNA promoter initiation regions of diploid wheats contain the sequences, which differ from the evolutionary conserved TATAGT A GG (+1 is underlined) motif of monocots. The transient expression assay in wheat protoplasts confirmed that cloned sequences are active promoters. Deletion analysis showed that the promoter sequence localized between −113 and +15 (relative to +1) is enough to direct RNA polymerase I-dependent transcription.