scholarly journals Crystal structure and functional analysis of human C1ORF123

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5377 ◽  
Author(s):  
Siti Nurulnabila A. Rahaman ◽  
Jastina Mat Yusop ◽  
Zeti-Azura Mohamed-Hussein ◽  
Wan Mohd Aizat ◽  
Kok Lian Ho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sequence Similarity ◽  
Physiological Role ◽  
Zinc Ion ◽  
Internal Symmetry ◽  
Chromosome 1 ◽  
Zinc Binding ◽  
Binding Motif ◽  
Functional Studies

Proteins of the DUF866 superfamily are exclusively found in eukaryotic cells. A member of the DUF866 superfamily, C1ORF123, is a human protein found in the open reading frame 123 of chromosome 1. The physiological role of C1ORF123 is yet to be determined. The only available protein structure of the DUF866 family shares just 26% sequence similarity and does not contain a zinc binding motif. Here, we present the crystal structure of the recombinant human C1ORF123 protein (rC1ORF123). The structure has a 2-fold internal symmetry dividing the monomeric protein into two mirrored halves that comprise of distinct electrostatic potential. The N-terminal half of rC1ORF123 includes a zinc-binding domain interacting with a zinc ion near to a potential ligand binding cavity. Functional studies of human C1ORF123 and its homologue in the fission yeast Schizosaccharomyces pombe (SpEss1) point to a role of DUF866 protein in mitochondrial oxidative phosphorylation.

scholarly journals Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation

2018 ◽  
Vol 116 (2) ◽  
pp. 528-533 ◽  
Author(s):  
Ivan B. Lomakin ◽  
Sergey E. Dmitriev ◽  
Thomas A. Steitz
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Translation Initiation ◽  
Zinc Ion ◽  
Zinc Binding ◽  
Ion Binding ◽  
Binding Interface ◽  
Zinc Binding Site ◽  
Reading Frames ◽  
Zinc Ion Binding

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR’s MCT-1–binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

scholarly journals The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tibisay Guevara ◽  
Hagen Körschgen ◽  
Anna Cuppari ◽  
Carlo Schmitz ◽  
Michael Kuske ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Physiological Role ◽  
Zinc Ion ◽  
Terminal Region ◽  
Elephant Trunk ◽  
Regular Secondary Structure ◽  
Mechanism Of Inhibition ◽  
Human Fertility ◽  
Complex Crystal Structure

Abstract Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

scholarly journals The zinc-binding motif of human RECQ5β suppresses the intrinsic strand-annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme

2008 ◽  
Vol 412 (3) ◽  
pp. 425-433 ◽  
Author(s):  
Hua Ren ◽  
Shuo-Xing Dou ◽  
Xing-Dong Zhang ◽  
Peng-Ye Wang ◽  
Radhakrishnan Kanagaraj ◽  
...  
Keyword(s):  
Structural Similarity ◽  
Zinc Binding ◽  
Binding Motif ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
The Novel ◽  
Quantitative Measurements ◽  
Strand Annealing ◽  
Zinc Binding Motif

RecQ family helicases, functioning as caretakers of genomic integrity, contain a zinc-binding motif which is highly conserved among these helicases, but does not have a substantial structural similarity with any other known zinc-finger folds. In the present study, we show that a truncated variant of the human RECQ5β helicase comprised of the conserved helicase domain only, a splice variant named RECQ5α, possesses neither ATPase nor DNA-unwinding activities, but surprisingly displays a strong strand-annealing activity. In contrast, fragments of RECQ5β including the intact zinc-binding motif, which is located immediately downstream of the helicase domain, exhibit much reduced strand-annealing activity but are proficient in DNA unwinding. Quantitative measurements indicate that the regulatory role of the zinc-binding motif is achieved by enhancing the DNA-binding affinity of the enzyme. The novel intramolecular modulation of RECQ5β catalytic activity mediated by the zinc-binding motif may represent a universal regulation mode for all RecQ family helicases.

scholarly journals A high-content screen profiles cytotoxic microRNAs in pediatric and adult glioblastoma cells and identifies miR-1300 as a potent inducer of cytokinesis failure

2019 ◽  
Author(s):  
Marjorie Boissinot ◽  
Henry King ◽  
Matthew Adams ◽  
Julie Higgins ◽  
Thomas A. Ward ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Ectopic Expression ◽  
Physiological Role ◽  
Mrna Translation ◽  
Glioblastoma Cells ◽  
Potent Inducer ◽  
Functional Studies ◽  
Key Points ◽  
Megakaryocyte Differentiation

AbstractBackgroundMicroRNAs play an important role in the regulation of mRNA translation, and have therapeutic potential in cancer and other diseases.MethodsTo profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen using a synthetic oligonucleotide library representing all known human microRNAs in adult and pediatric GBM cells. Bio-informatics analysis were used to refine this list and the top seven microRNAs were validated in a larger panel of cells by flow-cytometry, and RTqPCR. The downstream mechanism of the strongest and most consistent candidate was investigated by siRNAs, 3’UTR luciferase assays and Western Blotting.ResultsOur screen identified ∼100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 expressing cells and characterized the mechanism of action as cytokinesis failure followed by apoptosis, which was observed in an extended GBM cell panel including two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In glioblastoma cells, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target of miR-1300. ECT2 siRNA phenocopied the effects of miR-1300, and its overexpression led to a significant rescue of miR-1300 induced binucleation.ConclusionMiR-1300 was identified as a novel regulator of endomitosis with translatable potential for therapeutic application. The datatasets will be a resource for the neuro-oncology community.Key points (2 or 3 key points 85 characters plus spaces each)70% of cytotoxic microRNAs were shared between adult and pediatric glioblastoma cellsMiR-1300 expression is restricted to endomitosis within megakaryocyte differentiationMiR-1300’s ectopic expression is a potent and promising therapeutic tool in cancerImportance of StudyPrevious functional studies of microRNAs involved in the regulation of glioblastoma cell proliferation and/or survival have focused on adult glioblastoma alone and are restricted to only a few microRNAs at a time. Our study provides the first encompassing landscape of potent cytotoxic microRNAs in pediatric and adult glioblastoma.Not only, does our data provide an invaluable resource for the research community but it also revealed that 70% of microRNAs with significant cytotoxicity were shared by adult and pediatric cells. Finally, we identified and characterized the previously undescribed role of microRNA-1300 in the tight regulation of megakaryocyte differentiation into platelets and how, when expressed outside of this context, miR-1300 consistently causes cytokinesis failure followed by apoptosis, and thus represents a powerful cytotoxic tool with potential for translation towards therapeutic applications.

scholarly journals Genome-wide identification and expression profiling of invertase gene family for abiotic stresses tolerance in Poncirus trifoliata

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Bachar Dahro ◽  
Yue Wang ◽  
Ahmed Alhag ◽  
Chunlong Li ◽  
Dayong Guo ◽  
...  
Keyword(s):  
Gene Family ◽  
Abiotic Stresses ◽  
Family Members ◽  
Physiological Role ◽  
Poncirus Trifoliata ◽  
High Accumulation ◽  
Functional Studies ◽  
Genome Wide ◽  
Cold Tolerant

Abstract Background Sucrose (Suc) hydrolysis is directly associated with plants tolerance to multiple abiotic stresses. Invertase (INV) enzymes irreversibly catalyze Suc degradation to produce glucose (Glc) and fructose (Frc). However, genome-wide identification and function of individual members of the INV gene family in Poncirus trifoliata or its Citrus relatives in response to abiotic stresses are not fully understood. Results In this report, fourteen non-redundant PtrINV family members were identified in P. trifoliata including seven alkaline/neutral INV genes (PtrA/NINV1–7), two vacuolar INV genes (PtrVINV1–2), and five cell wall INV isoforms (PtrCWINV1–5). A comprehensive analysis based on the biochemical characteristics, the chromosomal location, the exon–intron structures and the evolutionary relationships demonstrated the conservation and the divergence of PtrINVs. In addition, expression analysis of INV genes during several abiotic stresses in various tissues indicated the central role of A/NINV7 among INV family members in response to abiotic stresses. Furthermore, our data demonstrated that high accumulation of Suc, Glc, Frc and total sugar contents were directly correlated with the elevated activities of soluble INV enzymes in the cold-tolerant P. trifoliata, C. ichangensis and C. sinensis, demonstrating the potential role of soluble INV enzymes for the cold tolerance of Citrus. Conclusions This work offered a framework for understanding the physiological role of INV genes and laid a foundation for future functional studies of these genes in response to abiotic stresses.

Probing the Mechanistic Role of Glutamate Residue in the Zinc-Binding Motif of Type A Botulinum Neurotoxin Light Chain†

Biochemistry ◽  
2000 ◽  
Vol 39 (9) ◽  
pp. 2399-2405 ◽  
Author(s):  
Li ◽  
Thomas Binz ◽  
Heiner Niemann ◽  
Bal Ram Singh
Keyword(s):  
Light Chain ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Zinc Binding ◽  
Binding Motif ◽  
Zinc Binding Motif

scholarly journals Co-SELECT reveals sequence non-specific contribution of DNA shape to transcription factor binding in vitro

2018 ◽  
Author(s):  
Soumitra Pal ◽  
Jan Hoinka ◽  
Teresa M. Przytycka
Keyword(s):  
Dna Binding ◽  
Sequence Similarity ◽  
Binding Motif ◽  
Sequence Motif ◽  
Shape Features ◽  
Specific Shape ◽  
Dna Shape ◽  
Specific Contribution

AbstractUnderstanding the principles of DNA binding by transcription factors (TFs) is of primary importance for studying gene regulation. Recently, several lines of evidence suggested that both DNA sequence and shape contribute to TF binding. However, the question if in the absence of any sequence similarity to the binding motif, DNA shape can still increase probability of binding was yet to be addressed.To address this challenge, we developed Co-SELECT, a computational approach to analyze the results of in vitro HT-SELEX experiments for TF-DNA binding. Specifically, the presence of motif-free sequences in late HT-SELEX rounds and their enrichment in weak binders allowed us to detect evidence for the role of DNA shape features in TF binding.Our approach revealed that, even in the absence of the sequence motif, TFs have propensity to weakly bind to DNA molecules enriched in specific shape features. Surprisingly, we also found that some properties of DNA shape contribute to promiscuous binding of all tested TF families. Strikingly, such promiscuously bound shapes correspond to the most frequent shape formed by the DNA. We propose that this promiscuous binding facilitates diffusing of TFs along the DNA molecule before it is locked in its binding site.

scholarly journals AMPK promotes Arf6 activation in a kinase-independent manner upon energy deprivation

2021 ◽  
Author(s):  
Kuan Jung S Chen ◽  
Jia-Wei Hsu ◽  
Fang-Jen S Lee
Keyword(s):  
Physiological Role ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Regulatory Domain ◽  
Nucleotide Exchange ◽  
Independent Manner ◽  
Invasion And Migration ◽  
And Migration ◽  
Energy Deprivation

AMP-activated protein kinase (AMPK) is a crucial cellular nutrient and energy sensor that maintains energy homeostasis. AMPK also governs cancer cell invasion and migration by regulating gene expression and activating multiple cellular signaling pathways. ADP-ribosylation factor 6 (Arf6) can be activated via nucleotide exchange by guanine nucleotide exchange factors (GEFs), and its activation also regulates tumor invasion and migration. By studying GEF-mediated Arf6 activation, we elucidated that AMPK functions as a noncanonical GEF for Arf6 in a kinase-independent manner. Moreover, by examining the physiological role of the AMPK-Arf6 axis, we determined that AMPK activates Arf6 upon glucose starvation and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) treatment. We further identified the binding motif in the C-terminal regulatory domain of AMPK that is responsible for promoting Arf6 activation and thus inducing cell migration and invasion. These findings reveal a noncanonical role of AMPK in which its C-terminal regulatory domain serves as a GEF for Arf6 during energy deprivation.

The crystal structure of human α1-microglobulin reveals a potential haem-binding site

2012 ◽  
Vol 445 (2) ◽  
pp. 175-182 ◽  
Author(s):  
Winfried Meining ◽  
Arne Skerra
Keyword(s):  
Binding Site ◽  
Sequence Similarity ◽  
Physiological Role ◽  
Terminal Segment ◽  
Human Blood Plasma ◽  
Binding Motif ◽  
Side Chains ◽  
Complement Protein ◽  
Loop Region ◽  
Prostaglandin D Synthase

We describe the 2.3 Å (1 Å=0.1 nm) X-ray structure of α1m (α1-microglobulin), an abundant protein in human blood plasma, which reveals the β-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 which is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the β-barrel, as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni2+-binding site together with a crystallographic neighbour. The closest structural relatives of α1m are the complement protein component C8γ, the L-prostaglandin D synthase and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, α1m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI–MS (electrospray ionization MS) provide evidence for a chromophore bound to the recombinant α1m, also known as ‘yellow/brown lipocalin’. However, the three side chains of Lys92, Lys118 and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well-known Cys–Pro haem-binding motif indicates the presence of a haem-binding site within the loop region of α1m, which explains previous biochemical findings and supports a physiological role in haem scavenging, as well as redox-mediated detoxification.

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