ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

Acetylation of WCC is dispensable for the core circadian clock but differentially regulates acute light responses in Neurospora.

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) has been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is controlled mainly by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Combination of NKG2A and PD-1 Blockade Improves Radiotherapy Response in Radioresistant Tumors.

Radiotherapy (RT) is commonly employed to treat solid tumors. Immune checkpoint blockade of programmed cell death protein 1 (PD-1) and CTLA-4 improves survival in RT patients, yet many fail to respond to combination therapy. Natural killer group 2 (NKG2) family receptors, particularly inhibitory NKG2A and activating NKG2D, have emerged as promising therapeutic targets to improve antitumor T cell responses; thus, we examined how these receptors and their ligands (Qa-1b and retinoic acid early inducible 1 [Rae-1], respectively) regulate the RT response in C57BL/6 mice bearing syngeneic B16F10 melanoma and MC38 colorectal adenocarcinoma tumors. RT (15 Gy) transiently reduced B16F10 tumor burden, whereas MC38 tumors exhibited durable response to RT. Intratumoral NK and CD8 T cells expressed NKG2A and NKG2D in both models, which was unaltered by RT. In vitro/in vivo RT increased tumor/stromal cell Qa-1b and Rae-1 expression in both models, especially B16F10 tumors, but IFN-γ stimulation induced both Qa-1b and Rae-1 only in B16F10 tumors. NKG2A/Qa-1b inhibition alone did not improve RT response in either model, but combined RT and NKG2A/PD-1 blockade improved survival in the B16F10 model. Depletion experiments indicate that the triple therapy efficacy is CD8 T cell-dependent with negligible NK cell contribution. RNA sequencing of CD8 T cells from triple therapy-treated B16F10 tumors showed increased proliferative capacity compared with RT and PD-1 blockade alone. Our work demonstrates that RT modulates NKG2A ligand expression, which inhibits RT-induced T cell responses in tumors that fail to respond to combined RT and PD-1 blockade. These results provide a rationale for combining NKG2A blockade with immune checkpoint blockade therapies and RT to improve clinical response.

Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA-containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7-family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

High-Density Polyethylene Custom Focusing Lenses for High-Resolution Transient Terahertz Biomedical Imaging Sensors.

Transient terahertz time-domain spectroscopy (THz-TDS) imaging has emerged as a novel non-ionizing and noninvasive biomedical imaging modality, designed for the detection and characterization of a variety of tissue malignancies due to their high signal-to-noise ratio and submillimeter resolution. We report our design of a pair of aspheric focusing lenses using a commercially available lens-design software that resulted in about 200 × 200-µm2 focal spot size corresponding to the 1-THz frequency. The lenses are made of high-density polyethylene (HDPE) obtained using a lathe fabrication and are integrated into a THz-TDS system that includes low-temperature GaAs photoconductive antennae as both a THz emitter and detector. The system is used to generate high-resolution, two-dimensional (2D) images of formalin-fixed, paraffin-embedded murine pancreas tissue blocks. The performance of these focusing lenses is compared to the older system based on a pair of short-focal-length, hemispherical polytetrafluoroethylene (TeflonTM) lenses and is characterized using THz-domain measurements, resulting in 2D maps of the tissue refractive index and absorption coefficient as imaging markers. For a quantitative evaluation of the lens effect on the image resolution, we formulated a lateral resolution parameter, R2080, defined as the distance required for a 20-80% transition of the imaging marker from the bare paraffin region to the tissue region in the same image frame. The R2080 parameter clearly demonstrates the advantage of the HDPE lenses over TeflonTM lenses. The lens-design approach presented here can be successfully implemented in other THz-TDS setups with known THz emitter and detector specifications.

Evaluation of Quantification and Normalization Strategies for Phosphoprotein Phosphatase Affinity Proteomics: Application to Breast Cancer Signaling.

Accurate quantification of proteomics data is essential for revealing and understanding biological signaling processes. We have recently developed a chemical proteomic strategy termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) to investigate endogenous phosphoprotein phosphatase (PPP) dephosphorylation signaling. Here, we compare the robustness and reproducibility of status quo quantification methods for optimal performance and ease of implementation. We then apply PIB-MS to an array of breast cancer cell lines to determine differences in PPP signaling between subtypes. Breast cancer, a leading cause of cancer death in women, consists of three main subtypes: estrogen receptor-positive (ER+), human epidermal growth factor receptor two positive (HER2+), and triple-negative (TNBC). Although there are effective treatment strategies for ER+ and HER2+ subtypes, tumors become resistant and progress. Furthermore, TNBC has few targeted therapies. Therefore, there is a need to identify new approaches for treating breast cancers. Using PIB-MS, we distinguished TNBC from non-TNBC based on subtype-specific PPP holoenzyme composition. In addition, we identified an increase in PPP interactions with Hippo pathway proteins in TNBC. These interactions suggest that phosphatases in TNBC play an inhibitory role on the Hippo pathway and correlate with increased expression of YAP/TAZ target genes both in TNBC cell lines and in TNBC patients.

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa.

Lung infections caused by antibiotic-resistant strains of Pseudomonas aeruginosa are difficult to eradicate in immunocompromised hosts such as those with cystic fibrosis. We previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AECs) deliver microRNA let-7b-5p to P. aeruginosa to suppress biofilm formation and increase sensitivity to beta-lactam antibiotics. In this study, we show that EVs secreted by AECs transfer multiple distinct short RNA fragments to P. aeruginosa that are predicted to target the three subunits of the fluoroquinolone efflux pump MexHI-OpmD, thus increasing antibiotic sensitivity. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%), and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S ribosomal RNA (rRNA) external transcribed spacer that was transferred to P. aeruginosa by EVs reduced planktonic growth of P. aeruginosa in the presence of ciprofloxacin, reduced the minimum inhibitory concentration of P. aeruginosa for ciprofloxacin by over 50%, and significantly reduced protein levels of both MexH and OpmD. In conclusion, an rRNA fragment secreted by AECs in EVs that targets the fluoroquinolone efflux pump MexHI-OpmD downregulated these proteins and increased the ciprofloxacin sensitivity of P. aeruginosa. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with ciprofloxacin-resistant P. aeruginosa infections.NEW & NOTEWORTHY Human RNA fragments transported in extracellular vesicles interfere with Pseudomonas aeruginosa drug efflux pumps. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Proteomics for the Discovery of Clinical Delirium Biomarkers: A Systematic Review of Major Studies.

Delirium represents a significant health care burden, diagnosed in more than 2 million elderly Americans each year. In the surgical population, delirium remains the most common complication among elderly patients, and is associated with longer hospital stays, higher costs of care, increased mortality, and functional impairment. The pathomechanism of disease is poorly understood, with current diagnostic approaches somewhat subjective and arbitrary, and definitive diagnostic biomarkers are currently lacking. Despite the recent interest in delirium research, biomarker discovery for it remains new. Most attempts to discover biomarkers are targeted studies that seek to assess the involvement of one or more members of a focused panel of candidates in delirium. For a more unbiased, system-biology view, we searched literature from Medical Literature Analysis and Retrieval System Online (MEDLINE), Cochrane Central, Web of Science, SCOPUS, and Dimensions between 2016 and 2021 for untargeted proteomic discovery studies for biomarkers of delirium conducted on human geriatric subjects. Two reviewers conducted an independent review of all search results and resolved discordance by consensus. From an overall search of 1172 publications, 8 peer-reviewed studies met our defined inclusion criteria. The 370 unique perioperative biomarkers identified in these reports are enriched in pathways involving activation of the immune system, inflammatory response, and the coagulation cascade. The most frequently identified biomarker was interleukin-6 (IL-6). By reviewing the distribution of protein biomarker candidates from these studies, we conclude that a panel of proteins, rather than a single biomarker, would allow for discriminating delirium cases from noncases. The paucity of hypothesis-generating studies in the peer-reviewed literature also suggests that a system-biology view of delirium pathomechanisms has yet to fully emerge.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Harnessing the Immunological Effects of Radiation to Improve Immunotherapies in Cancer.

Ionizing radiation (IR) is used to treat 50% of cancers. While the cytotoxic effects related to DNA damage with IR have been known since the early 20th century, the role of the immune system in the treatment response is still yet to be fully determined. IR can induce immunogenic cell death (ICD), which activates innate and adaptive immunity against the cancer. It has also been widely reported that an intact immune system is essential to IR efficacy. However, this response is typically transient, and wound healing processes also become upregulated, dampening early immunological efforts to overcome the disease. This immune suppression involves many complex cellular and molecular mechanisms that ultimately result in the generation of radioresistance in many cases. Understanding the mechanisms behind these responses is challenging as the effects are extensive and often occur simultaneously within the tumor. Here, we describe the effects of IR on the immune landscape of tumors. ICD, along with myeloid and lymphoid responses to IR, are discussed, with the hope of shedding light on the complex immune stimulatory and immunosuppressive responses involved with this cornerstone cancer treatment. Leveraging these immunological effects can provide a platform for improving immunotherapy efficacy in the future.

The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI.

Iron is an essential nutrient for plants, but excess iron is toxic due to its catalytic role in the formation of hydroxyl radicals. Thus, iron uptake is highly regulated and induced only under iron deficiency. The mechanisms of iron uptake in roots are well characterized, but less is known about how plants perceive iron deficiency. We show that a basic helix-loop-helix (bHLH) transcription factor Upstream Regulator of IRT1 (URI) acts as an essential part of the iron deficiency signaling pathway in Arabidopsis thaliana The uri mutant is defective in inducing Iron-Regulated Transporter1 (IRT1) and Ferric Reduction Oxidase2 (FRO2) and their transcriptional regulators FER-like iron deficiency-induced transcription factor (FIT) and bHLH38/39/100/101 in response to iron deficiency. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) reveals direct binding of URI to promoters of many iron-regulated genes, including bHLH38/39/100/101 but not FIT While URI transcript and protein are expressed regardless of iron status, a phosphorylated form of URI only accumulates under iron deficiency. Phosphorylated URI is subject to proteasome-dependent degradation during iron resupply, and turnover of phosphorylated URI is dependent on the E3 ligase BTS. The subgroup IVc bHLH transcription factors, which have previously been shown to regulate bHLH38/39/100/101, coimmunoprecipitate with URI mainly under Fe-deficient conditions, suggesting that it is the phosphorylated form of URI that is capable of forming heterodimers in vivo. We propose that the phosphorylated form of URI accumulates under Fe deficiency, forms heterodimers with subgroup IVc proteins, and induces transcription of bHLH38/39/100/101 These transcription factors in turn heterodimerize with FIT and drive the transcription of IRT1 and FRO2 to increase Fe uptake.

Bladder Cancer Extracellular Vesicles Elicit a CD8 T Cell-Mediated Antitumor Immunity.

Tumor-derived extracellular vesicles (TEVs) play crucial roles in mediating immune responses, as they carry and present functional MHC-peptide complexes that enable them to modulate antigen-specific CD8+ T-cell responses. However, the therapeutic potential and immunogenicity of TEV-based therapies against bladder cancer (BC) have not yet been tested. Here, we demonstrated that priming with immunogenic Extracellular Vesicles (EVs) derived from murine MB49 BC cells was sufficient to prevent MB49 tumor growth in mice. Importantly, antibody-mediated CD8+ T-cell depletion diminished the protective effect of MB49 EVs, suggesting that MB49 EVs elicit cytotoxic CD8+ T-cell-mediated protection against MB49 tumor growth. Such antitumor activity may be augmented by TEV-enhanced immune cell infiltration into the tumors. Interestingly, MB49 EV priming was unable to completely prevent, but significantly delayed, unrelated syngeneic murine colon MC-38 tumor growth. Cytokine array analyses revealed that MB49 EVs were enriched with pro-inflammatory factors that might contribute to increasing tumor-infiltrating immune cells in EV-primed MC-38 tumors. These results support the potential application of TEVs in personalized medicine, and open new avenues for the development of adjuvant therapies based on patient-derived EVs aimed at preventing disease progression.

A HAD family phosphatase CSP-6 regulates the circadian output pathway in Neurospora crassa.

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δcsp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δwhi-2 mutant phenotypes resemble Δcsp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δcsp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δcsp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output.

Microspheres Encapsulating Immunotherapy Agents Target the Tumor-Draining Lymph Node in Pancreatic Ductal Adenocarcinoma.

INTRODUCTION: The tumor-draining lymph node (TDLN) plays a role in tumor immunity. Intratumorally administered microspheres (MS) that encapsulate immunomodulatory agents have emerged as a treatment strategy capable of causing profound changes in the tumor microenvironment (TME) and eliciting potent antitumor effects. We hypothesized that local delivery of MS to the TME may also drain to and therefore target the TDLN to initiate antitumor immune responses. METHODS: Fluorescent MS were injected into orthotopically implanted murine pancreatic tumors, and tissues were examined by whole-mount microscopy and imaging flow cytometry. The role of the TDLN was investigated for mice treated with intratumoral interleukin-12 (IL-12)-encapsulated MS in combination with stereotactic body radiotherapy (SBRT) by cytokine profile and TDLN ablation. RESULTS: Fluorescent AF-594 MS delivered intratumorally were detected in the tumor, peritumoral lymphatics, and the TDLN 2 h after injection. Phagocytic cells were observed with internalized fluorescent MS. SBRT + IL-12 MS-induced upregulation of Th1 and antitumor factors IL-12, IFN-γ, CXCL10, and granzyme B in the TDLN, and excision of the TDLN partially abrogated treatment efficacy. CONCLUSIONS: Our results demonstrate that intratumorally administered MS not only target the TME, but also drain to the TDLN. Furthermore, MS encapsulated with a potent antitumor cytokine, IL-12, induce an antitumor cytokine profile in the TDLN, which is essential for treatment efficacy.

Phosphatidylinositol-(3,4,5)-Trisphosphate Induces Phagocytosis of Nonmotile Pseudomonas aeruginosa.

Pathogenic bacteria that establish chronic infections in immunocompromised patients frequently undergo adaptation or selection for traits that are advantageous for their growth and survival. Clinical isolates of Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterial pathogen, exhibit a temporal transition from a motile to a nonmotile phenotype through loss of flagellar motility during the course of chronic infection. This progressive loss of motility is associated with increased resistance to both antibiotic and immune clearance. We have previously shown that loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and in vivo and fails to activate the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent phagocytic pathway. Therefore, we tested the hypothesis that clearance of phagocytosis-resistant bacteria could be induced by exogenously pretreating innate immune cells with the Akt-activating molecule phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). Here, we demonstrate that PIP3 induces the uptake of nonmotile P. aeruginosa by primary human neutrophils >25-fold, and this effect is phenocopied with the use of murine phagocytes. However, surprisingly, mechanistic studies revealed that the induction of phagocytosis by PIP3 occurs because polyphosphoinositides promote bacterial binding by the phagocytes rather than bypassing the requirement for PI3K. Moreover, this induction was selective since the uptake of other nonmotile Gram-negative, but not Gram-positive, bacteria can also be induced by PIP3 Since there is currently no treatment that effectively eradicates chronic P. aeruginosa infections, these findings provide novel insights into a potential methodology by which to induce clearance of nonmotile pathogenic bacteria and into the endogenous determinants of phagocytic recognition of P. aeruginosa.

A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles.

Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.

Myc protein is stabilized by suppression of a novel E3 ligase complex in cancer cells.

Rapid Myc protein turnover is critical for maintaining basal levels of Myc activity in normal cells and a prompt response to changing growth signals. We characterize a new Myc-interacting factor, TRPC4AP (transient receptor potential cation channel, subfamily C, member 4-associated protein)/TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein), which is the receptor for a DDB1 (damage-specific DNA-binding protein 1)-CUL4 (Cullin 4) E3 ligase complex for selective Myc degradation through the proteasome. TRPC4AP/TRUSS binds specifically to the Myc C terminus and promotes its ubiquitination and destruction through the recognition of evolutionarily conserved domains in the Myc N terminus. TRPC4AP/TRUSS suppresses Myc-mediated transactivation and transformation in a dose-dependent manner. Finally, we found that TRPC4AP/TRUSS expression is strongly down-regulated in most cancer cell lines, leading to Myc protein stabilization. These studies identify a novel pathway targeting Myc degradation that is suppressed in cancer cells.

Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells.

Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2(-/-) NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321-1331.

Offline pentafluorophenyl (PFP)-RP prefractionation as an alternative to high-pH RP for comprehensive LC-MS/MS proteomics and phosphoproteomics.

Technological advances in liquid chromatography and tandem mass spectrometry (LC-MS/MS) have enabled comprehensive analyses of proteins and their post-translational modifications from cell culture and tissue samples. However, sample complexity necessitates offline prefractionation via a chromatographic method that is orthogonal to online reversed-phase high-performance liquid chromatography (RP-HPLC). This additional fractionation step improves target identification rates by reducing the complexity of the sample as it is introduced to the instrument. A commonly employed offline prefractionation method is high pH reversed-phase (Hi-pH RP) chromatography. Though highly orthogonal to online RP-HPLC, Hi-pH RP relies on buffers that interfere with electrospray ionization. Thus, samples that are prefractionated using Hi-pH RP are typically desalted prior to LC-MS/MS. In the present work, we evaluate an alternative offline prefractionation method, pentafluorophenyl (PFP)-based reversed-phase chromatography. Importantly, PFP prefractionation results in samples that are dried prior to analysis by LC-MS/MS. This reduction in sample handling relative to Hi-pH RP results in time savings and could facilitate higher target identification rates. Here, we have compared the performances of PFP and Hi-pH RP in offline prefractionation of peptides and phosphopeptides that have been isolated from human cervical carcinoma (HeLa) cells. Given the prevalence of isobaric mass tags for peptide quantification, we evaluated PFP chromatography of peptides labeled with tandem mass tags. Our results suggest that PFP is a viable alternative to Hi-pH RP for both peptide and phosphopeptide offline prefractionation.