Biology subjects

Woods, N. T.

Publications and source records attributed to Woods, N. T..

5 recordsLinked to original sources

bioRxiv2026

Integrative Proteomic Analysis Implicates Inhibition of Intracellular Protein Trafficking in Therapy-Induced Migrastasis in Prostate Cancer

BackgroundDysregulated cell migration leading to metastasis remains the primary cause of cancer-related mortality. It has been challenging to understand how cells regulate migration. We have previously created the first selective inhibitor of cell migration, KBU2046. Here, we use it as a probe to identify regulatory processes. MethodsMetastatic and primary human prostate cancer cells were treated for different times and at different concentrations with KBU2046. Immunofluorescent microscopy examined protein localization in cells. Label-free mass spectrometry (MS) was performed on total cell proteins, Tandem Mass Tag (TMT) labeling MS was used on membrane fractions, and temporal phosphoproteomic profiling. Results were analyzed with a suite of bioinformatic tools. ResultsKBU2046-induced migrastasis is associated with the accumulation of activated integrin {beta}1 into focal adhesions. Whole-cell proteomics demonstrated suppression of processes that mediate intracellular protein trafficking and increases in mitochondrial energy-generation signatures. Evaluation of the membrane fraction identified increases in membrane repair and maintenance processes and decreases in those that drive motility. Temporal- and concentration-dependent phosphoproteomic profiling revealed that KBU2046 initiates a dynamic, cascading sequence of transient signaling waves rather than a static block. ConclusionsKBU2046-induced migrastasis appears to operate through spatial decoupling rather than structural degradation. By restricting the intracellular trafficking machinery required for receptor recycling, KBU2046 limits focal adhesion turnover, providing a correlative framework to inhibit metastatic dissemination independent of direct cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/736165v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1efd74dorg.highwire.dtl.DTLVardef@1202106org.highwire.dtl.DTLVardef@581ed3org.highwire.dtl.DTLVardef@14fdcfb_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG

cancer biology↗

bioRxiv2022

SMALL MOLECULE ACTIVATORS OF B56-PP2A RESTORE 4E-BP EXPRESSION AND FUNCTION TO SUPPRESS CAP-DEPENDENT TRANSLATION IN CANCER CELLS

Dysregulation of cap-dependent translation is a hallmark of cancer, with key roles in supporting the transformed phenotype. The eIF4E binding proteins (4E-BP1, 2, 3) are major negative regulators of cap-dependent translation that are inactivated in tumors through inhibitory phosphorylation by oncogenic kinases (e.g., mTOR) or by downregulation. Previous studies from our group and others have linked tumor suppressive PP2A family serine/threonine phosphatases to activation of 4E-BP1. Here, we leveraged novel small molecule activators of PP2A (SMAPs) (e.g., DT-061, DT-1154) that are being developed as antitumor agents to (a) explore the role of a subset of B56-PP2As in regulation of 4E-BP activity, and (b) to evaluate the potential of B56-PP2A reactivation for restoring translation control in tumor cells. We show that SMAPs promote PP2A-dependent hypophosphorylation of 4E-BP1/4EBP2 in the presence of active upstream inhibitory kinases (mTOR, ERK, AKT), supporting a role for B56-PP2As as 4E-BP phosphatases. Unexpectedly, DT-061 also led to robust PP2A-dependent upregulation of 4E-BP1, but not 4E-BP2 or 4E-BP3. Cap-binding assays and eIF4E immunoprecipitation showed that SMAP/B56-PP2A blocks the formation of the eIF4F translation initiation complex. Bicistronic reporter assays that directly measure cap-dependent translation activity confirmed the translational consequences of these effects. siRNA knockdown pointed to B56-PP2A as a mediator of SMAP effects on 4E-BPs, although B56{beta}- and/or B56{varepsilon}-PP2A may also play a role. 4E-BP1 upregulation involved ATF4-dependent transcription of the 4E-BP1 gene (EIF4EBP1) and the effect was partially dependent on TFE3/TFEB transcription factors. Thus, B56-PP2A orchestrates a translation repressive program involving transcriptional induction and hypophosphorylation of 4E-BP1, highlighting the potential of PP2A-based therapeutic strategies for restoration of translation control in cancer cells.

cancer biology↗

bioRxiv2022

Functional Requirements for a Samd14-Capping Protein Complex in Stress Erythropoiesis

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system and promotes stress-dependent c-Kit signaling. However, the mechanism underlying Samd14s role in stress erythropoiesis is unknown. We identified a protein- protein interaction between Samd14 and the - and {beta} heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP {beta} subunit increased erythroid maturation in ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/c-kit signaling in CD71med spleen erythroid precursors. Given the roles of c-Kit signaling in hematopoiesis and Samd14 in c-Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.

cell biology↗

bioRxiv2019

E3 Ligase UBR5 HECT domain mutations in lymphoma control maturation of B cells via alternative splicing

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5 with the majority of mutations within the HECT domain of the protein which can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B cell maturation and activation we generated a conditional mutant disrupting UBR5s C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and up-regulation of proteins associated with mRNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B cell maturation by regulating alternative splicing of key transcripts during B cell development and suggests UBR5 mutations may promote mantle cell lymphoma initiation.\n\nKEY POINTSO_LIUtilizing a novel mouse model mimicking MCL patient mutations, the loss of UBR5s HECT domain causes alterations in B cell development.\nC_LIO_LIUBR5 mutations lead to stabilization of UBR5 and aberrant splicing.\nC_LI

immunology↗

bioRxiv2019

Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole in Chlamydia trachomatis Infected Human Cells

As an obligate intracellular pathogenic bacterium, C. trachomatis develops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. An in vivo understanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 in Chlamydia trachomatis L2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.\n\nAuthor summaryMany intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound \"bacteria containing vacuole\" (BCV) that, in most cases, prevents association with the lysosome. Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotin in vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane site in vivo in the context of infection.

microbiology↗