Biology subjects

Gonzalez, D.

Publications and source records attributed to Gonzalez, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Cardiac Effects of a Novel pH-Insensitive Sodium-Calcium Exchanger Mouse

BACKGROUNDCardiac sodium-calcium exchange (NCX1) is the dominant calcium (Ca) efflux mechanism in cardiomyocytes and is strongly regulated by pH. However, the role of NCX1 pH sensitivity in normal cardiac function is unknown. METHODSWe used CRISPR/Cas9 to produce a pH-resistant NCX1 mouse by replacing the histidine at position 165 of NCX1 with an alanine (H165A). Hearts were studied using echocardiography and ECG. RNA and protein expression levels were assessed using qPCR and Western blotting. Isolated ventricular cardiomyocytes were loaded with Ca indicators and patch clamped to record intracellular Ca transients and membrane current and voltage. RESULTSH165A mice live into adulthood with slightly reduced LV systolic function, normal heart rate and shortened QT interval. Both male and female animals exhibit reduced growth, but females eventually reach normal body weight. In patch clamped myocytes, NCX current (INCX) evoked by voltage ramps was reduced by 35% (at +80 mV). Lowering pHi to 6.5 using Na-Acetate had no effect on INCX in H165A myocytes, whereas the same intervention in wildtype (WT) inhibited INCX by 69% (at +80 mV, p<0.01). There was no change in H165A ventricular cardiomyocyte Ca transients measured with fura-2 AM. However, action potential duration was reduced 68%, consistent with the shorter QT interval. This coincided with a 37% reduction in L-type Ca current and increased expression of repolarizing K+ channels. H165A mice are also resistant to ischemia/reperfusion injury. CONCLUSIONSThe H165A mutation attenuates pH regulation of NCX1 in mice, is associated with reduced growth and accelerates cardiac repolarization without compromising excitation-contraction coupling. The mutation also confers cardioprotection. The H165A mouse is the first evidence that pH regulation of NCX1 affects cardiac physiology and is a potential model for studying the role of NCX1 pH-sensitivity on both physiological and pathophysiological cardiac function.

biophysics↗

bioRxiv2022

Spatiotemporal coordination of stem cell behavior following alveolar injury

Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 (AT2) cells act as stem cells and can replace both themselves and alveolar type 1 cells (AT1); however, the complex orchestration of AT2 stem cell activity following lung injury is poorly understood owing to the inability of tracking individual stem cells and their dynamic behavior over time. Here, we apply live time lapse imaging to ex vivo mouse precision cut lung slice (PCLS) culture and in vivo mouse lung to track individual GFP-labeled AT2 cells following induction of alveolar injury by bleomycin. We observe highly dynamic movement of AT2 cells, including migration within and between alveoli. To map the dynamic evolution of AT2 cell behavior, we introduce Live Cell Encoder (LCE-PHATE), a novel method for converting static snapshots from time lapse imaging into single points representative of entire, dynamic cellular trajectories. Applying LCE-PHATE, we observe the emergence of at least three distinct morphokinetic AT2 cell states associated with AT2 stem cell injury response. Finally, small molecule-based inhibition of Rho-associated protein kinase (ROCK) pathway significantly reduced motility of AT2 stem cells following injury and reduced expression of Krt8, a marker of intermediate progenitor cells. Together, our results uncover motility of alveolar stem cells as a new injury response mechanism in the lung and reveal properties of stem cell motility at high cellular resolution.

cell biology↗

bioRxiv2022

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of keratin-10 nascent RNA, which marks the onset of stem cell differentiation, we find that keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

genetics↗

bioRxiv2022

A Zur-mediated transcriptional regulation of the zinc export system

The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of Pseudomonas aeruginosa. The transcription of its many Zn transporters is therefore tightly regulated by a known set of transcription factors involved in either the import or the export of Zn. In this work, we show that the Zur protein, a well-known repressor of Zn import, plays a dual role and functions in both import and export processes. In a situation of Zn excess, Zur represses Zn entry, but also activates the transcription of czcR, a positive regulator of the Zn export system. To achieve this, Zur binds at two sites, located by DNA footprinting in the region downstream the czcR transcription start site. In agreement with this regulation, a delay in induction of the efflux system is observed in the absence of Zur and Zn resistance is affected. The discovery of this regulation highlights a new role of Zur as global regulator of Zn homeostasis in P. aeruginosa disclosing an important link between Zur and zinc export.

microbiology↗

bioRxiv2022

Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation

Tissue regeneration and maintenance rely on coordinated stem cell behaviors. This orchestration can be impaired by oncogenic mutations leading to tissue architecture disruption and ultimately cancer formation. However, it is still largely unclear how oncogenes perturb stem cells functions to break tissue architecture. Here, we used intravital imaging and a novel signaling reporter to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking the same hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner. This tissue architecture abnormality is linked with a spatial dysregulation of stem cell proliferation as well as abnormal migration during hair follicle growth. By using a reporter mouse that allows us to capture real-time ERK signal dynamics at the single cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts the pulsatile ERK signal fluctuation in the stem cells into sustained activation. Furthermore, by combining drug treatment with longitudinal imaging, we demonstrated that temporary inhibiting ERK signal reverts the KrasG12D-induced tissue deformation, suggesting that sustained ERK activation leads to tissue architecture disruption in Kras mutant hair follicles. Altogether, our work suggests that oncogenic mutations induce tissue abnormalities when spatiotemporally specific conditions are met, which allows mutant stem cells to disturb local cell coordination through altering dynamic signal communications.

cancer biology↗

bioRxiv2022

Aerosolized Desert Dusts Collected Near a Drying Lake and Pulmonary Inflammation in Mice: Implications for Environmental Exposures and Asthma

BackgroundA high incidence of asthma is prevalent among residents near the Salton Sea, a large inland terminal lake in southern California. This arid region has high levels of ambient particulate matter (PM); yet while high PM levels are often associated with asthma in many environments, it is possible that the rapidly retreating lake may contribute components with a specific role in promoting asthma symptoms. ObjectivesOur hypothesis is that asthma may be higher in residents closest to the Salton Sea due to chronic exposures to playa dust. Playa emissions may be concentrating dissolved material from the lake, with microbial components capable of inducing pulmonary innate immune responses. Such inflammatory responses may contribute to the development of asthma-like symptoms in residents. To test this hypothesis, we used a mouse model of aerosol exposures to assess the effects of playa dust. MethodsFrom dust collected around the Salton Sea region, aqueous extracts were used to generate aerosols, which were injected into an environmental chamber for mouse exposure studies. We compared the effects of exposure to Salton Sea aerosols, as well as to known immunostimulatory reference materials. Acute 48-hour and chronic 7-day exposures were compared, with lungs analyzed for inflammatory cell recruitment and gene expression. ResultsDust from sites nearest to the Salton Sea triggered lung neutrophil inflammation that was stronger at 48-hours but reduced at 7-days. This acute inflammatory profile and kinetics resembled the response to innate immune ligands LTA and LPS while distinct from the classic allergic response to Alternaria. ConclusionLung inflammatory responses to Salton Sea dusts are similar to acute innate immune responses, raising the possibility that microbial components are entrained in the dust, promoting inflammation. This effect highlights the health risks at drying terminal lakes from inflammatory components in dust emissions from exposed lakebed.

pharmacology and toxicology↗

bioRxiv2022

Ecology shapes the cyclic dominance games between Escherichia coli strains

Evolutionary game theory has provided various models to explain the coexistence of competing strategies, one of which is the rock-paper-scissors (RPS) game. A system of three Escherichia coli strains--a toxin-producer, a resistant, and a sensitive--has become a classic experimental model for studying RPS games. Previous experimental and theoretical studies, however, often ignored the influence of ecological factors such as nutrients and toxin dynamics on the evolutionary game dynamics. In this work, we combine experiments and modeling to study how these factors affect competition dynamics. Using 3D-printed mini-bioreactors, we tracked the frequency of the three strains in different culturing media and under different flow regimes. Although our experimental system fulfilled the requirements of cyclic dominance, we did not observe clear cycles or long-term coexistence between strains. We found that both nutrients and flow rates strongly impacted population dynamics. In our simulations, we explicitly modeled the release, removal and diffusion of toxin. We showed that the amount of toxin that is retained in the system is a simple indicator that can predict competition outcomes across broad parameter space. Moreover, our simulation results suggest that high rates of toxin diffusion might have prevented cyclic patterns from emerging in our experimental system.

evolutionary biology↗

bioRxiv2022

Autocrine Sfrp1 inhibits lung fibroblast invasion during transition to injury induced myofibroblasts

Fibroblast to myofibroblast conversion is a major driver of tissue remodeling in organ fibrosis. Several distinct lineages of fibroblasts support homeostatic tissue niche functions, yet, specific activation states and phenotypic trajectories of fibroblasts during injury and repair have remained unclear. Here, we combined spatial transcriptomics, longitudinal single-cell RNA-seq and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. We discovered a transitional fibroblast state characterized by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1+ cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1+ transitional fibroblasts and Cthrc1+ myofibroblasts. Tgf{beta}1 downregulated Sfrp1 in non-invasive transitional cells and induced their switch to an invasive Cthrc1+ myofibroblast identity. Finally, using loss of function studies we showed that autocrine Sfrp1 directly inhibits fibroblast invasion by regulating the RhoA pathway. In summary, our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies Sfrp1 as an autocrine inhibitor of fibroblast invasion during early stages of fibrogenesis.

cell biology↗

bioRxiv2022

Intracellular Calcium links Milk Stasis to Lysosome Dependent Cell Death during Mammary Gland Involution

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase- independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2- 4 hours of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6 and TGF{beta}3, all of which appear to be upregulated by increased intracellular calcium. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis. This is the result of increased TGF{beta} signaling and inhibition of cell cycle progression. Finally, we demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3, a process which also appears to be mediated by TGF{beta} signaling. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome- mediated cell death.

cell biology↗

bioRxiv2022

Design and construction of 3D-printed devices to investigate active and passive bacterial dispersal on hydrated surfaces

To disperse in water-unsaturated environments, such as the soil, bacteria rely on the availability and structure of water films forming on biotic and abiotic surfaces, and, especially, along fungal mycelia. Dispersal along such "fungal highways" is driven both by mycelial physical properties and by interactions between bacteria and fungi. To understand the role of abiotic elements, we designed and 3D-printed two devices establishing stable liquid films that support bacteria dispersal in the absence of biotic interactions. The thickness of the liquid film determined the presence of hydraulic flow capable of carrying non-motile cells. In the absence of flow, only motile cells can disperse in the presence of an energy source. Non-motile cells could not disperse autonomously without flow, but dispersed when co-inoculated with motile cells. By teasing apart the abiotic and biotic dimensions, these 3D-printed devices will stimulate further research on microbial dispersal in soil and other water-unsaturated environments.

microbiology↗

bioRxiv2022

An NKX-COUP-TFII genomic code for mucosal vascular addressins and organ morphogenesis

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules, providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define a genomic address code for targeted expression of mucosal vascular addressins and implicate NCCE in fundamental processes in cell specification and development.

immunology↗

bioRxiv2022

KaiC-like proteins improve stress resistance in environmental Pseudomonas species

KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologs of this protein are widespread among bacteria that are not known to have or need a circadian physiology. The function, interaction network, and mechanism of action of these KaiC homologs are still largely unknown. Here, we focus on KaiC-like proteins found in environmental Pseudomonas species. Using a bioinformatic approach, we describe the diversity and distribution of members of this protein family in the Pseudomonas genus and sketch, through comparative genomics, a conserved minimal interaction network comprising a histidine kinase and a response regulator. We then characterize experimentally the only KaiC homolog present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in sulfur uptake and alternative carbon source utilization in P. protegens. As expected, it physically interacts with its cognate histidine kinase and response regulator. Moreover, KaiC homologs are phosphorylated at one (P. putida) or two (P. protegens) sites and KaiC phosphorylation patterns change over time; however, in Pseudomonas species, changes in KaiC phosphorylation are driven by the age of the culture rather than by circadian cues as is the case in Cyanobacteria. In this study, through thorough bioinformatic and experimental analyses, we shed light onto the functional diversification and evolution of a unique protein family, diversely involved in bacterial rhythmic interactions with their environment. By so doing, we present a striking example of a protein whose general purpose is conserved in evolution, but whose molecular mechanics and participation in bacterial physiology can change dramatically across species.

microbiology↗

bioRxiv2022

Spatial scales of competition and a growth-motility tradeoff interact to determine bacterial coexistence

1.The coexistence of competing species is a long-lasting puzzle in evolutionary ecology research. Despite abundant experimental evidence showing that the opportunity for coexistence decreases as niche overlap increases between species, bacterial species and strains competing for the same resources are commonly found across diverse spatially heterogeneous habitats. We thus hypothesized that the spatial scale of competition may play a key role in determining bacterial coexistence, and interact with other mechanisms that promote coexistence, including a growth-motility tradeoff. To test this hypothesis, we let two Pseudomonas putida strains compete at local and regional scales by inoculating them either in a mixed droplet or in separate droplets in the same Petri dish, respectively. We also created conditions that allow the bacterial strains to disperse across abiotic or fungal hyphae networks. We found that competition at the local scale led to competitive exclusion while regional competition promoted coexistence. When competing in the presence of dispersal networks, the growth-motility tradeoff promoted coexistence only when the strains were inoculated in separate droplets. Our results provide a mechanism by which existing laboratory data suggesting competitive exclusion at a local scale is reconciled with the widespread coexistence of competing bacterial strains in complex natural environments with dispersal.

ecology↗

bioRxiv2021

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse {beta}-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug- resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers. IMPACT STATEMENTDisruption of disulfide bond formation sensitizes resistant Gram- negative bacteria expressing {beta}-lactamases and mobile colistin resistance enzymes to currently available antibiotics.

microbiology↗

bioRxiv2021

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage ([≥] 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

biochemistry↗

bioRxiv2021

Proteomic Organellar Mapping Reveals Modulation of Peroxisomes by HIV-1

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new, unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses we found that classifier performance in this system was organelle-dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine (SVM) learning for mitochondrial and ER proteins, but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, BANDLE, on SVM-classified data. Comparative BANDLE analysis of cells induced to express the wild-type viral genome vs. cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as TCR signaling components and coatomer complex. Lastly, we found that SVM classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.

cell biology↗

bioRxiv2021

Sequence-unrelated long noncoding RNAs converged to modulate the activity of conserved epigenetic machineries across kingdoms

RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Here, we showed that in addition to the PRC1-component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrated that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we showed that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter. Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation, and reveal that evolutionary unrelated lncRNAs may exert similar functions across kingdoms.

plant biology↗

bioRxiv2021

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpus itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 required PTPN23 but not the retromer subunits. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23. Author SummaryVpu triggers the degradation or mis-localization of proteins important to the hosts immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the proteins location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpus presence at the limiting membranes of multi-vesicular bodies.

microbiology↗