scholarly journals ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

2020 ◽  
Vol 133 (18) ◽  
pp. jcs243477 ◽  
Author(s):  
Franziska Eck ◽  
Santosh Phuyal ◽  
Matthew D. Smith ◽  
Manuel Kaulich ◽  
Simon Wilkinson ◽  
...  
Keyword(s):  
Binding Site ◽  
Genome Editing ◽  
Lipid Droplet ◽  
Protein Complexes ◽  
Major Drawback ◽  
Binding Partner ◽  
Atg Genes ◽  
Interaction Proteomics

ABSTRACTWhile studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.

scholarly journals ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

2020 ◽  
Author(s):  
Franziska Eck ◽  
Manuel Kaulich ◽  
Christian Behrends
Keyword(s):  
Functional Analysis ◽  
Lipid Droplet ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Major Drawback ◽  
Interacting Protein ◽  
Binding Partner ◽  
Lipid Droplet Formation ◽  
Atg Genes ◽  
Interaction Proteomics

AbstractWhile studies of ATG genes in knockout models led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3/GABARAP proteins are still poorly understood. A major drawback for their understanding is that the available interactome data was largely acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was exemplarily employed to map endogenous GABARAPL2 protein complexes in response to autophagic modulation using interaction proteomics. This approach identified the ER transmembrane protein and lipid droplet biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. Through this interaction, the GABARAPL2-interacting protein and UFM1-activating enzyme UBA5 becomes anchored at the ER membrane. Functional analysis unveiled ACSL3 and lipid droplet formation as novel regulators of the enigmatic UFM1 conjugation pathway.

scholarly journals Identification of critical residues in the colicin E9 DNase binding region of the Im9 protein

1997 ◽  
Vol 323 (3) ◽  
pp. 823-831 ◽  
Author(s):  
Michael J. OSBORNE ◽  
Russell WALLIS ◽  
Kit-Yi LEUNG ◽  
Glyn WILLIAMS ◽  
Lu-Yun LIAN ◽  
...  
Keyword(s):  
Binding Site ◽  
Protein Complexes ◽  
Dissociation Rate ◽  
15N Nmr ◽  
Binding Region ◽  
Nmr Studies ◽  
Immunity Protein ◽  
Wide Range ◽  
Protein Dissociation ◽  
Colicin E9

1H–15N NMR studies, in conjunction with mutagenesis experiments, have been used to delineate the DNase-binding surface of the colicin E9 inhibitor protein Im9 (where Im stands for immunity protein). Complexes were formed between the 15 kDa unlabelled E9 DNase domain and the 9.5 kDa Im9 protein uniformly labelled with 15N. Approx. 90% of the amide resonances of the bound Im9 were assigned and spectral parameters obtained from 1H–15N heteronuclear single quantum coherence (HSQC) spectra were compared with those for the free Im9 assigned previously. Many of the amide resonances were shifted on complex formation, some by more than 2 p.p.m. in the 15N dimension and more than 0.5 p.p.m. in the 1H dimension. Most of the strongly shifted amides are located on the surfaces of two of the four helices, helix II and helix III. Whereas helix II had already been identified through genetic and biochemical investigations as an important determinant of biological specificity, helix III had not previously been implicated in binding to the DNase. To test the robustness of the NMR-delineated DNase-binding site, a selection of Im9 alanine mutants were constructed and their dissociation rate constants from E9 DNase-immunity protein complexes quantified by radioactive subunit exchange kinetics. Their off-rates correlated well with the NMR perturbation analysis; for example, residues that were highly perturbed in HSQC experiments, such as residues 34 (helix II) and 54 (helix III), had a marked effect on the DNase–immunity protein dissociation rate when replaced by alanine. The NMR and mutagenesis data are consistent with a DNase-binding region on Im9 composed of invariant residues in helix III and variable residues in helix II. The relationship of this binding site model to the wide range of affinities (Kd values in the range 10-4 to 10-16 M) that have been measured for cognate and non-cognate colicin DNase–immunity protein interactions is discussed.

scholarly journals Review of rationale and progress toward targeting cyclin-dependent kinase 2 (CDK2) for male contraception†

2020 ◽  
Vol 103 (2) ◽  
pp. 357-367
Author(s):  
Erik B Faber ◽  
Nan Wang ◽  
Gunda I Georg
Keyword(s):  
Cell Cycle ◽  
Binding Site ◽  
Knockout Mice ◽  
Male Contraception ◽  
Cyclin Dependent Kinase ◽  
Compensatory Mechanisms ◽  
Binding Partner ◽  
Male Contraceptive ◽  
Clinical Stages

Abstract Cyclin-dependent kinase 2 (CDK2) is a member of the larger cell cycle regulating CDK family of kinases, activated by binding partner cyclins as its name suggests. Despite its canonical role in mitosis, CDK2 knockout mice are viable but sterile, suggesting compensatory mechanisms for loss of CDK2 in mitosis but not meiosis. Here, we review the literature surrounding the role of CDK2 in meiosis, particularly a cyclin-independent role in complex with another activator, Speedy 1 (SPY1). From this evidence, we suggest that CDK2 could be a viable nonhormonal male contraceptive target. Finally, we review the literature of pertinent CDK2 inhibitors from the preclinical to clinical stages, mostly developed to treat various cancers. To date, there is no potent yet selective CDK2 inhibitor that could be repurposed as a contraceptive without appreciable off-target toxicity. To achieve selectivity for CDK2 over closely related kinases, developing compounds that bind outside the conserved adenosine triphosphate-binding site may be necessary.

scholarly journals Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum

2019 ◽  
Vol 47 (22) ◽  
pp. 11574-11588 ◽  
Author(s):  
Wieteke Anna Maria Hoeijmakers ◽  
Jun Miao ◽  
Sabine Schmidt ◽  
Christa Geeke Toenhake ◽  
Sony Shrestha ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Complexes ◽  
Human Malaria Parasite ◽  
Phd Finger ◽  
Finger Protein ◽  
Binding Preference ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Eukaryotic Organisms ◽  
Interaction Proteomics

Abstract Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.

The 5S RNA – protein complex from yeast: a model for the evolution and structure of the eukaryotic ribosome

1982 ◽  
Vol 60 (4) ◽  
pp. 490-496 ◽  
Author(s):  
Ross N. Nazar ◽  
Makoto Yaguchi ◽  
Gordon E. Willick
Keyword(s):  
Binding Site ◽  
Protein Complex ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Basic Structure ◽  
5S Rna ◽  
Chemical Studies ◽  
Physical And Chemical ◽  
Rna Protein Complexes

The ribosomal 5S RNA – protein complex appears to be an excellent model for studies on the evolution and structure of ribosomes. In eukaryotes this complex is composed of two components, the 5S rRNA and a single ribosomal protein which in yeast has a molecular weight of about 38 000. The primary protein-binding site is located in the 3′-end region of the 5S RNA together with a small portion of the 5′ end. The primary RNA-binding site appears to be situated in the C-terminal end of the protein (YL3 in yeast) but the binding specificity requires other structural elements in the N-terminal half of the molecule. When compared with prokaryotic 5S RNA – protein complexes, various physical and chemical studies suggest that the basic structure and interactions have been conserved in the course of evolution, but that the single larger eukaryotic 5S RNA binding protein has evolved through a fusion of genes for the multiple 5S RNA binding proteins in prokaryotes.

scholarly journals Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

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