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Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains. [ABSTRACT FROM AUTHOR]

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance. Author summary: Piperacillin/tazobactam and amikacin are common antibiotic choices for treating bloodstream infections, which are potentially lethal and predominantly caused by the bacterium Escherichia coli. By studying a large collection of bloodstream E. coli we show that dual resistance to piperacillin/tazobactam and amikacin is unexpectedly common. We explain this by showing that two resistance genes are circulating widely as part of a transposon. We show that genetic changes in the transposon, most commonly an increase in the number of copies of the transposon in the genome, are required to confer co-resistance to both antibiotics. Hence we have identified an important, emerging, mobile genetic element with the potential to compromise therapy by two commonly used antibiotics for bloodstream infection. We also show how this knowledge might be used to improve the sensitivity of tests for piperacillin/tazobactam resistance by integrating the results of amikacin resistance testing, reducing treatment failure and sparing last resort antibiotics. [ABSTRACT FROM AUTHOR]

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly. [ABSTRACT FROM AUTHOR]

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well‐established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole‐cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross‐polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery. [ABSTRACT FROM AUTHOR]

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection. Daly, Danson and colleagues employ a multi-omic approach in neuroglioma cells to characterise endolysosomal dysfunction caused by perturbation of the evolutionarily conserved Retromer complex, highlighting Retromer's neuroprotective function. [ABSTRACT FROM AUTHOR]

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60 (also known as DYNC2I1) and the DYNC2H1-DYNC2LI1 dimer from dynein-2, and IFT54 (also known as TRAF3IP1) and IFT57 from IFT-B contribute to the dynein-2-IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that Nterminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection. [ABSTRACT FROM AUTHOR]

Metastasis and recurrence of breast cancer remain major causes of patient mortality, and there is an ongoing need to identify new therapeutic targets relevant to tumor invasion. Protein disulfide isomerase A3 (PDIA3) is a disulfide oxidoreductase and isomerase of the endoplasmic reticulum that has known extracellular substrates and has been correlated with aggressive breast cancers. We show that either prior PDIA3 inhibition by the disulfide isomerase inhibitor 16F16 or depletion of heparin-binding proteins strongly reduces the activity of conditioned medium (CM) of MDA-MB-231 human breast cancer cells to support promigratory cell spreading and F-actin organization by newly adherent MDA-MB-231 cells. Quantitative proteomics to investigate effects of 16F16 inhibition on heparin-binding proteins in the CM of MDA-MB-231 cells identified 80 proteins reproducibly decreased at least twofold (at q ≤ 0.05) after 16F16 treatment. By Gene Ontology analysis, many of these have roles in extracellular matrix (ECM) structure and function and cell adhesion; ribosomal proteins that also correlate with extracellular vesicles were also identified. Protein-protein interaction analysis showed that many of the extracellular proteins have known network interactions with each other. The predominant types of disulfide-bonded domains in the extracellular proteins contained β-hairpin folds, with the knottin fold the most common. From human breast cancer data sets, the extracellular proteins were found to correlate specifically with the basal subtype of breast cancer and their high expression in tumors correlated with reduced distant metastasis- free survival. These data provide new evidence that PDIA3 may be a relevant therapeutic target to alter properties of the ECM-associated microenvironment in basal breast cancer. [ABSTRACT FROM AUTHOR]

The matricellular glycoprotein thrombospondin-1 (TSP1) has complex roles in the extracellular matrix (ECM) and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulfide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalize in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3-/- mouse embryo fibroblasts (Pdia3-/- MEFs), led to alterations in the composition of cell-derived extracellular matrix, involving changed abundance of fibronectin and TSP1, was correlated with reduced cell spreading, altered organization of F-actin, and reduced focal adhesions. These cellular phenotypes of Pdia3-/- MEFs were normalized by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEFs. Rescue depended on PDIA3 activity in WTMEFs and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEFs and Pdia3-/- MEFs identified multiple ECM and growth factor proteins to be downregulated in the CM of Pdia3-/- MEFs. Of these, cell communication network 2 (CCN2) was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3-/- MEFs and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, proadhesive microenvironment, with implications for PDIA3 as a translational target. [ABSTRACT FROM AUTHOR]

The emergence of antimicrobial resistance (AMR) represents a significant health and economic challenge worldwide. The slow pace of antibacterial discovery necessitates strategies for optimal use of existing agents, including effective diagnostics able to drive informed prescribing; and development of alternative therapeutic strategies that go beyond traditional small‐molecule approaches. Thus, the development of novel probes able to target bacteria for detection and killing, and that can pave the way to effective theranostic strategies, is of great importance. Here we demonstrate that metal‐free green‐emitting fluorescent carbon dots (FCDs) synthesized from glucosamine·HCl and m‐phenylenediamine, and featuring 2,5‐deoxyfructosazine on a robust amorphous core, can label both Gram‐positive (Staphylococcus aureus) and Gram‐negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) bacterial pathogens within 10 minutes of exposure. Moreover, effective killing of Gram‐positive and ‐negative bacteria can be induced by combining FCD treatment with irradiation by LED light in the visible range. Cell‐based, electron microscopy and tandem mass tag (TMT) proteomic experiments indicate that FCD administration in combination with LED exposure gives rise to local heating, ROS production, and membrane‐ and DNA‐damage, suggesting multiple routes to FCD‐mediated bacterial killing. Our data identify FCDs as materials that combine facile synthesis from low‐cost precursors with labeling and light‐dependent killing of clinically important bacterial species, and that thus warrant further exploration as the potential bases for novel theranostics. [ABSTRACT FROM AUTHOR]