Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN’s UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.

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The GET pathway serves to activate Atg32-mediated mitophagy by ER targeting of the Ppg1-Far complex

10.1101/2021.09.20.461067 ◽

2021 ◽

Author(s):

Mashun Onishi ◽

Koji Okamoto

Keyword(s):

Posttranslational Modification ◽

Scaffold Protein ◽

Mitochondrial Proteins ◽

Aaa Atpase ◽

Selective Autophagy ◽

Er Targeting ◽

Additional Loss

AbstractMitophagy removes defective or superfluous mitochondria via selective autophagy. In yeast, the pro-mitophagic protein Atg32 localizes to the mitochondrial surface and interacts with the scaffold protein Atg11 to promote degradation of mitochondria. Although Atg32-Atg11 interactions are thought to be stabilized by Atg32 phosphorylation, how this posttranslational modification is regulated remains obscure. Here we show that cells lacking the guided entry of tail-anchored proteins (GET) pathway exhibit reduced Atg32 phosphorylation and Atg32-Atg11 interactions, which can be rescued by additional loss of the ER-resident Ppg1-Far complex, a multi-subunit phosphatase negatively acting in mitophagy. In GET-deficient cells, Ppg1-Far is predominantly localized to mitochondria. An artificial ER anchoring of Ppg1-Far in GET-deficient cells significantly ameliorates defects in Atg32-Atg11 interactions and mitophagy. Moreover, disruption of GET and Msp1, an AAA-ATPase that extracts non-mitochondrial proteins localized to the mitochondrial surface, elicits synthetic defects in mitophagy. Collectively, we propose that the GET pathway mediates ER targeting of Ppg1-Far, thereby preventing dysregulated suppression of mitophagy activation.

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Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Applied and Environmental Microbiology ◽

10.1128/aem.67.10.4678-4684.2001 ◽

2001 ◽

Vol 67 (10) ◽

pp. 4678-4684 ◽

Cited By ~ 32

Author(s):

Henrik Wernérus ◽

Janne Lehtiö ◽

Tuula Teeri ◽

Per-Åke Nygren ◽

Stefan Ståhl

Keyword(s):

Metal Binding ◽

Binding Capacity ◽

Surface Display ◽

Surface Proteins ◽

Chimeric Proteins ◽

Phage Display Technology ◽

Binding Domains ◽

Combinatorial Protein Engineering ◽

Display Technology ◽

Cellulose Binding

ABSTRACT Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reeseicellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux

Molecular and Cellular Biology ◽

10.1128/mcb.00392-17 ◽

2017 ◽

Vol 38 (1) ◽

Cited By ~ 38

Author(s):

Laura Pontano Vaites ◽

Joao A. Paulo ◽

Edward L. Huttlin ◽

J. Wade Harper

Keyword(s):

Quantitative Proteomics ◽

Ectopic Expression ◽

Late Endosome ◽

Autophagic Flux ◽

Systematic Analysis ◽

Selective Autophagy ◽

Regulatory Circuits ◽

Guanine Exchange Factor ◽

Interaction Proteomics ◽

Autophagosome Maturation

ABSTRACT Selective autophagy and macroautophagy sequester specific organelles/substrates or bulk cytoplasm, respectively, inside autophagosomes as cargo for delivery to lysosomes. The mammalian ATG8 orthologues (MAP1LC3A/B/C and GABARAP/L1/L2) are ubiquitin (UB)-like proteins conjugated to the autophagosome membrane and are thought to facilitate cargo receptor recruitment, vesicle maturation, and lysosomal fusion. To elucidate the molecular functions of the ATG8 proteins, we engineered cells lacking genes for each subfamily as well as all six mammalian ATG8s. Loss of GABARAPs alone attenuates autophagic flux basally and in response to macroautophagic or selective autophagic stimuli, including parkin-dependent mitophagy, and cells lacking all ATG8 proteins accumulate cytoplasmic UB aggregates, which are resolved following ectopic expression of individual GABARAPs. Autophagosomes from cells lacking GABARAPs had reduced lysosomal content by quantitative proteomics, consistent with fusion defects, but accumulated regulators of late endosome (LE)/autophagosome maturation. Through interaction proteomics of proteins accumulating in GABARAP/L1/L2-deficient cells, we identified C18orf8/RMC1 as a new subunit of the CCZ1-MON1 RAB7 guanine exchange factor (GEF) that positively regulates RAB7 recruitment to LE/autophagosomes. This work defines unique roles for GABARAP and LC3 subfamilies in macroautophagy and selective autophagy and demonstrates how analysis of autophagic machinery in the absence of flux can identify new regulatory circuits.

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BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS

Science Advances ◽

10.1126/sciadv.aav8463 ◽

2019 ◽

Vol 5 (8) ◽

pp. eaav8463 ◽

Cited By ~ 6

Author(s):

Ruth Röck ◽

Johanna E. Mayrhofer ◽

Omar Torres-Quesada ◽

Florian Enzler ◽

Andrea Raffeiner ◽

...

Keyword(s):

Resistance Mechanisms ◽

Tumor Formation ◽

Braf V600e ◽

Braf Inhibitors ◽

Braf Mutations ◽

Drug Induced ◽

Kinase Activation ◽

Binding Domains ◽

Intramolecular Communication ◽

Sequential Formation

Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation–containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

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Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase

Biochemical Journal ◽

10.1042/bj2690261 ◽

1990 ◽

Vol 269 (1) ◽

pp. 261-264 ◽

Cited By ~ 55

Author(s):

L M A Ferreira ◽

A J Durrant ◽

J Hall ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Activity ◽

Specific Activity ◽

Binding Capacity ◽

Spatial Separation ◽

Crystalline Cellulose ◽

Conserved Sequence ◽

Binding Domains ◽

Catalytically Active ◽

Cellulose Binding ◽

Derivatives Of

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA′), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.

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The EGFL7-ITGB3-KLF2 axis enhances survival of multiple myeloma in preclinical models

Blood Advances ◽

10.1182/bloodadvances.2019001002 ◽

2020 ◽

Vol 4 (6) ◽

pp. 1021-1037 ◽

Cited By ~ 1

Author(s):

Yousef Salama ◽

Andries Hendrik Heida ◽

Kazuaki Yokoyama ◽

Satoshi Takahashi ◽

Koichi Hattori ◽

...

Keyword(s):

Multiple Myeloma ◽

Neutralizing Antibodies ◽

Plasma Cells ◽

Standard Of Care ◽

Primary Diagnosis ◽

Kinase Activation ◽

Amplification Loop ◽

Novel Strategy

Abstract Angiogenic factors play a key role in multiple myeloma (MM) growth, relapse, and drug resistance. Here we show that malignant plasma cells (cell lines and patient-derived MM cells) express angiocrine factor EGF like-7 (EGFL7) mRNA and protein. MM cells both produced EGFL7 and expressed the functional EGFL7 receptor integrin β 3 (ITGB3), resulting in ITGB3 phosphorylation and focal adhesion kinase activation. Overexpression of ITGB3 or EGFL7 enhanced MM cell adhesion and proliferation. Intriguingly, ITGB3 overexpression upregulated the transcription factor Krüppel-like factor 2 (KLF2), which further enhanced EGFL7 transcription in MM cells, thereby establishing an EGFL7-ITGB3-KLF2-EGFL7 amplification loop that supports MM cell survival and proliferation. EGFL7 expression was found in certain plasma cells of patients with refractory MM and of patients at primary diagnosis. NOD.CB17-Prkdc<scid>/J mice transplanted with MM cells showed elevated human plasma EGFL7 levels. EGFL7 knockdown in patient-derived MM cells and treatment with neutralizing antibodies against EGFL7 inhibited MM cell growth in vitro and in vivo. We demonstrate that the standard-of-care MM drug bortezomib upregulates EGFL7, ITGB3, and KLF2 expression in MM cells. Inhibition of EGFL7 signaling in synergy with BTZ may provide a novel strategy for inhibiting MM cell proliferation.

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Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

10.1101/2021.07.19.452535 ◽

2021 ◽

Author(s):

Vinay Eapen ◽

Sharan Swarup ◽

Melisa J Hoyer ◽

Joao A Paulo ◽

J Wade Harper

Keyword(s):

Hela Cells ◽

Quantitative Proteomics ◽

Central Component ◽

Regulatory Factor ◽

Future Studies ◽

Selective Autophagy ◽

Ubiquitin Binding ◽

Necessary And Sufficient ◽

Induced Neurons ◽

Lysosomal Proteins

Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both Hela cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

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Serine ADP-ribosylation reversal by the hydrolase ARH3

eLife ◽

10.7554/elife.28533 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 72

Author(s):

Pietro Fontana ◽

Juan José Bonfiglio ◽

Luca Palazzo ◽

Edward Bartlett ◽

Ivan Matic ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Posttranslational Modification ◽

Quantitative Proteomics ◽

Chromatin Regulation ◽

Adp Ribosylation ◽

Cellular Processes ◽

New Form ◽

Hydrolysis Of ◽

In Cells

ADP-ribosylation (ADPr) is a posttranslational modification (PTM) of proteins that controls many cellular processes, including DNA repair, transcription, chromatin regulation and mitosis. A number of proteins catalyse the transfer and hydrolysis of ADPr, and also specify how and when the modification is conjugated to the targets. We recently discovered a new form of ADPr that is attached to serine residues in target proteins (Ser-ADPr) and showed that this PTM is specifically made by PARP1/HPF1 and PARP2/HPF1 complexes. In this work, we found by quantitative proteomics that histone Ser-ADPr is reversible in cells during response to DNA damage. By screening for the hydrolase that is responsible for the reversal of Ser-ADPr, we identified ARH3/ADPRHL2 as capable of efficiently and specifically removing Ser-ADPr of histones and other proteins. We further showed that Ser-ADPr is a major PTM in cells after DNA damage and that this signalling is dependent on ARH3.

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The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

Microbial Cell Factories ◽

10.1186/s12934-021-01701-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Samira Ghaedmohammadi ◽

Gholamreza Ahmadian

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Bacillus Subtilis ◽

Binding Capacity ◽

Surface Display ◽

Protein A ◽

Production Costs ◽

Rabbit Serum ◽

Experimental Conditions ◽

Binding Domains

AbstractProtein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

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