scholarly journals The SUMO conjugation complex self-assembles into nuclear bodies independent of SIZ1 and COP1

2018 ◽  
Author(s):  
Magdalena J. Mazur ◽  
Mark Kwaaitaal ◽  
Manuel Arroyo Mateos ◽  
Francesca Maio ◽  
Ramachandra K. Kini ◽  
...  
Keyword(s):  
The Netherlands ◽  
Substrate Binding ◽  
E3 Ligase ◽  
Transcriptional Response ◽  
Binding Pocket ◽  
Nuclear Bodies ◽  
Acceptor Site ◽  
Night Time ◽  
Sumo Conjugation ◽  
Scientific Organisation

One sentence SummarySUMO conjugation activity causes formation of SUMO nuclear bodies, which strongly overlap with COP1 bodies thanks to a substrate-binding (VP) motif in the E3 ligase SIZ1 that acts as bridge protein.AbstractAttachment of the small ubiquitin-like modifier SUMO to substrate proteins modulates their turnover, activity or interaction partners. An unresolved question is how this SUMO conjugation activity concentrates the enzymes involved and the substrates into uncharacterized nuclear bodies (NBs). We here define the requirements for the formation of SUMO NBs and for their subsequent co-localisation with the master regulator of growth, the E3 ubiquitin ligase COP1. COP1 activity results in degradation of transcription factors, which primes the transcriptional response that underlies elongation growth induced by night-time and high ambient temperatures (skoto- and thermomorphogenesis, respectively). SUMO conjugation activity itself is sufficient to target the SUMO machinery into NBs. Co-localization of these bodies with COP1 requires besides SUMO conjugation activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SIZ1. We find that SIZ1 docks in the substrate-binding pocket of COP1 via two VP motifs - a known peptide motif of COP1 substrates. The data reveal that SIZ1 physically connects COP1 and SUMO conjugation activity in the same NBs that can also contain the blue-light receptors CRY1 and CRY2. Our findings thus suggest that sumoylation apparently coordinates COP1 activity inside these NBs; a mechanism that potentially explains how SIZ1 and SUMO both control the timing and amplitude of the high-temperature growth response. The strong co-localization of COP1 and SUMO in these NBs might also explain why many COP1 substrates are sumoylated.Funding informationThe Netherlands Scientific Organisation (ALW-VIDI grant 864.10.004 to HvdB) and the Topsector T&U program Better Plants for Demands (grant 1409-036 to HvdB), including the partnering breeding companies, supported this work; FM is financially supported by Keygene N.V. (The Netherlands).

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

scholarly journals Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

2019 ◽  
Vol 116 (39) ◽  
pp. 19552-19562 ◽  
Author(s):  
Justine Sitz ◽  
Sophie Anne Blanchet ◽  
Steven F. Gameiro ◽  
Elise Biquand ◽  
Tia M. Morgan ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Genomic Instability ◽  
E3 Ligase ◽  
Human Papillomaviruses ◽  
Double Strand Breaks ◽  
Nuclear Bodies ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa

2021 ◽  
Vol 77 (10) ◽  
pp. 1305-1316
Author(s):  
Yujing Chen ◽  
Haizhu Jia ◽  
Jianyu Zhang ◽  
Yakun Liang ◽  
Ruihua Liu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Class I ◽  
Polyamine Metabolism ◽  
Binding Assays ◽  
Starting Point ◽  
Family Activity ◽  
Living Organisms

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation–putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

scholarly journals Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1882
Author(s):  
Wei Xia ◽  
Yingguo Bai ◽  
Pengjun Shi
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Substrate Binding ◽  
Enzymatic Saccharification ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Substrate Affinity ◽  
Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

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