Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of ...pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.
Display omitted
•SdeA catalyzes atypical serine ubiquitination of substrates•Arginine phosphoribosylation of ubiquitin is a potent post-translational signal•Phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes•Phosphoribosylation of ubiquitin impairs several ubiquitin-dependent processes
Proteins can be ubiquitinated through a serine-linked phosphodiester bond independently of conventional E1 and E2 enzymes.
Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these ...tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549rDOX20) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-468r5-FU2000) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549rDOX20 and MDA-MB-468r5-FU2000 cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer.
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and ...partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
The 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.
Display omitted
•Phosphoribosyl serine ubiquitination can be reverted by DupA and DupB•DUP specifically binds to and cleaves PR-ubiquitin from serine on substrates•Catalytically inactive DupA mutants can capture PR-ubiquitinated proteins•PR ubiquitination on multiple ER structural proteins causes ER fragmentation
Shin et al. show that phosphoribosyl serine ubiquitination can be reversed by two deubiquitinases (DupA and DupB) from Legionella. Two DUPs specifically cleave PR-ubiquitin from serine on substrates. Catalytically inactive DupA-based proteomics approach reveals PR-ubiquitinated proteins and their roles in endoplasmic reticulum (ER) remodeling.
Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving ...post‐translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP‐L2 on surface‐exposed serine residues (LC3C S93 and S96; GABARAP‐L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP‐L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4‐mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP‐L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de‐conjugation of LC3C and GABARAP‐L2 to autophagosomes by TBK1‐mediated phosphorylation.
Synopsis
LC3C and GABARAP‐L2 processing by ATG4 is controlled by TBK1‐mediated phosphorylation. This regulatory mechanism prevents premature shedding of LC3C and GABARAP‐L2 from autophagosomes.
TBK1 phosphorylates autophagy modifiers LC3C and GABARAP‐L2 on surface‐exposed serine‐residues.
GABARAP‐L2 S87/88‐PO4 and LC3C S93/96‐PO4 impedes binding to the ATG4 protease.
Phosphorylated LC3C/GABARAP‐L2 are protected from ATG4‐mediated premature removal from autophagosomes.
LC3C and GABARAP‐L2 processing by ATG4 is controlled by TBK1‐mediated phosphorylation. This regulatory mechanism prevents premature shedding of LC3C and GABARAP‐L2 from autophagosomes.
Magnetotactic bacteria produce chains of complex membrane-bound organelles that direct the biomineralization of magnetic nanoparticles and serve for magnetic field navigation. These magnetosome ...compartments have recently emerged as a model for studying the subcellular organization of prokaryotic organelles. Previous studies indicated the presence of specific proteins with various functions in magnetosome biosynthesis. However, the exact composition and stoichiometry of the magnetosome subproteome have remained unknown.
In order to quantify and unambiguously identify all proteins specifically targeted to the magnetosome membrane of the Alphaproteobacterium Magnetospirillum gryphiswaldense, we analyzed the protein composition of several cellular fractions by semi-quantitative mass spectrometry. We found that nearly all genuine magnetosome membrane-integral proteins belong to a well-defined set of previously identified proteins encoded by gene clusters within a genomic island, indicating a highly controlled protein composition. Magnetosome proteins were present in different quantities with up to 120 copies per particle as estimated by correlating our results with available quantitative Western blot data. This high abundance suggests an unusually crowded protein composition of the membrane and a tight packing with transmembrane domains of integral proteins. Our findings will help to further define the structure of the organelle and contribute to the elucidation of magnetosome biogenesis.
Magnetosomes are one of the most complex bacterial organelles and consist of membrane-bounded crystals of magnetic minerals. The exact composition and stoichiometry of the associated membrane integral proteins are of major interest for a deeper understanding of prokaryotic organelle assembly; however, previous proteomic studies failed to reveal meaningful estimations due to the lack of precise and quantitative data, and the inherently high degree of accumulated protein contaminants in purified magnetosomes. Using a highly sensitive mass spectrometer, we acquired proteomic data from several cellular fractions of a magnetosome producing magnetotactic bacterium and developed a comparative algorithm to identify all genuine magnetosome membrane-integral proteins and to discriminate them from contaminants. Furthermore, by combining our data with previously published quantitative Western blot data, we were able to model the protein copy number and density within the magnetosome membrane. Our results suggest that the magnetosome membrane is specifically associated with a small subset of integral proteins that are tightly packed within the lipid layer. Our study provides by far the most comprehensive estimation of magnetosomal protein composition and stoichiometry and will help to elucidate the complex process of magnetosome biogenesis.
Display omitted
•First quantitative and unbiased assessment of the membrane-integral sub-proteome of bacterial organelles (magnetosomes).•A new algorithm distinguishes genuine magnetosome proteins from contaminants and identifies novel membrane constituents.•A specific subset of integral proteins is highly enriched and tightly packed within the magnetosome membrane.
The hydrothermal vent tubeworm
hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of ...physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that
symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.
Successful proteome analyses of highly dilute samples are strongly dependent on optimized workflows considering especially sample preparation prior to highly sensitive mass spectrometric analysis. ...Various methods are available for enrichment of proteome samples, each characterized by specific advantages and disadvantages limiting their general application as a method of choice. Here we suggest an optimized universal protocol ensuring reproducibility and effective enrichment of dilute samples by commercial affinity beads. By comparably assessing the performance of the new protocol with selected standard enrichment techniques, we show the seamless application of the enrichment in common mass spectrometry based proteomic workflows. Further, novel applications are suggested including a facile storage and shipping of desiccated, trapped proteome samples at ambient temperatures and usage of the affinity beads for gel-free proteomic approaches.
Autosomal dominant spinocerebellar ataxias (SCAs) are genetically heterogeneous neurological disorders characterized by cerebellar dysfunction mostly due to Purkinje cell degeneration. Here we show ...that AFG3L2 mutations cause SCA type 28. Along with paraplegin, which causes recessive spastic paraplegia, AFG3L2 is a component of the conserved m-AAA metalloprotease complex involved in the maintenance of the mitochondrial proteome. We identified heterozygous missense mutations in five unrelated SCA families and found that AFG3L2 is highly and selectively expressed in human cerebellar Purkinje cells. m-AAA-deficient yeast cells expressing human mutated AFG3L2 homocomplex show respiratory deficiency, proteolytic impairment and deficiency of respiratory chain complex IV. Structure homology modeling indicates that the mutations may affect AFG3L2 substrate handling. This work identifies AFG3L2 as a novel cause of dominant neurodegenerative disease and indicates a previously unknown role for this component of the mitochondrial protein quality control machinery in protecting the human cerebellum against neurodegeneration.
PDF
