Membrane-associated Factor Research Articles

ATP-binding cassette (ABC) transporters: structures and roles in bacterial pathogenesis.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporter systems are divided into importers and exporters that facilitate the movement of diverse substrate molecules across the lipid bilayer, against the concentration gradient. These transporters comprise two highly conserved nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Unlike ABC exporters, prokaryotic ABC importers require an additional substrate-binding protein (SBP) as a recognition site for specific substrate translocation. The discovery of a large number of ABC systems in bacterial pathogens revealed that these transporters are crucial for the establishment of bacterial infections. The existing literature has highlighted the roles of ABC transporters in bacterial growth, pathogenesis, and virulence. These roles include importing essential nutrients required for a variety of cellular processes and exporting outer membrane-associated virulence factors and antimicrobial substances. This review outlines the general structures and classification of ABC systems to provide a comprehensive view of the activities and roles of ABC transporters associated with bacterial virulence and pathogenesis during infection.

The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires' disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

The plasma membrane-associated transcription factor NAC091 regulates unfolded protein response in Arabidopsis thaliana

Adverse environmental stresses may cause the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), and the unfolded protein response (UPR) pathway is initiated to mitigate the ER stress. Previous studies demonstrate that NAC062, a plasma membrane-associated transcription factor, plays important roles in promoting cell survival under ER stress conditions in Arabidopsis thaliana. In this study, we identified another plasma membrane-associated transcription factor, NAC091 (also known as ANAC091/TIP), as an important UPR mediator. ER stress induces the expression of NAC091, which is mainly dependent on the ER stress regulators bZIP60 and bZIP28. In addition, NAC091 has transcriptional activation activity, and the truncated form of NAC091 devoid of the C-terminal transmembrane domain (TMD) forms a homodimer in the nucleus. Under ER stress conditions, NAC091 relocates from the plasma membrane to the nucleus and regulates the expression of canonical UPR genes involved in cell survival. Further, the loss-of-function mutant of NAC091 confers impaired ER stress tolerance. Together, these results reveal the important role of NAC091 in ER stress response in Arabidopsis, and demonstrate that NAC091 relays the ER stress signal from the plasma membrane to the nucleus to alleviate ER stress and promote cell survival in plants.

Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions. In silico analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5CTD) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δp5CTD and NTHi 3655Δp5, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δp5 CTD and NTHi 3655Δp5. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.

Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.

A virulence activator of a surface attachment protein in Burkholderia pseudomallei acts as a global regulator of other membrane-associated virulence factors.

Burkholderia pseudomallei (Bp), causing a highly fatal disease called melioidosis, is a facultative intracellular pathogen that attaches and invades a variety of cell types. We previously identified BP1026B_I0091 as a surface attachment protein (Sap1) and an essential virulence factor, contributing to Bp pathogenesis in vitro and in vivo. The expression of sap1 is regulated at different stages of Bp intracellular lifecycle by unidentified regulator(s). Here, we identified SapR (BP1026B_II1046) as a transcriptional regulator that activates sap1, using a high-throughput transposon mutagenesis screen in combination with Tn-Seq. Consistent with phenotypes of the Δsap1 mutant, the ΔsapR activator mutant exhibited a significant reduction in Bp attachment to the host cell, leading to subsequent decreased intracellular replication. RNA-Seq analysis further revealed that SapR regulates sap1. The regulation of sap1 by SapR was confirmed quantitatively by qRT-PCR, which also validated the RNA-Seq data. SapR globally regulates genes associated with the bacterial membrane in response to diverse environments, and some of the genes regulated by SapR are virulence factors that are required for Bp intracellular infection (e.g., type III and type VI secretion systems). This study has identified the complex SapR regulatory network and its importance as an activator of an essential Sap1 attachment factor.

A lily membrane-associated NAC transcription factor LlNAC014 is involved in thermotolerance via activation of the DREB2-HSFA3 module.

The NTL (NAC with transmembrane motif 1-like) transcription factors with a conserved transmembrane motif are members of the NAC family and are important in plant development and in response to stress. However, knowledge of their regulatory pathways is scarce, especially under heat stress. Here, we cloned and identified a novel lily (Lilium longiflorum) NTL gene, LlNAC014, that increases thermotolerance. High temperature repressed LlNAC014 expression but activated its protein. LlNAC014 contained a typical transmembrane motif at its far C-terminus and was normally located on membranes, but under heat stress it entered the nucleus as a transcription factor. LlNAC014 also has a transactivation domain at its C-terminus, and its active form, LlNAC014ΔC, could function as a trans-activator in both yeast and plant cells. LlNAC014ΔC overexpression in lily and Arabidopsis increased thermotolerance, and also caused growth defects; silencing LlNAC014 in lily decreased thermotolerance. LlNAC014ΔC could constitutively activate the heat stress response by inducing the expression of heat-responsive genes, some of which were dependent on the HSF (heat stress transcription factor) pathway. Further analysis showed that LlNAC014 was a direct regulator of the DREB2-HSFA3 module, and bound to the CTT(N7)AAG element in the promoters of LlHSFA3A, LlHSFA3B, and LlDREB2B to activate their expression. Thus, LlNAC014 increased thermotolerance by sensing high temperature and translocating to the nucleus to activate the DREB2-HSFA3 module.

Human B Cells Mediate Innate Anti-Cancer Cytotoxicity Through Concurrent Engagement of Multiple TNF Superfamily Ligands.

The essential innate immunity effector cells, natural killer and dendritic cells, express multiple plasma membrane-associated tumor necrosis factor (TNF) superfamily (TNFSF) ligands that, through simultaneous and synergistic engagement, mediate anti-cancer cytotoxicity. Here, we report that circulating B cells, mediators of adaptive humoral immunity, also mediate this innate anti-cancer immune mechanism. We show that resting human B cells isolated from peripheral blood induce apoptosis of, and efficiently kill a large variety of leukemia and solid tumor cell types. Single-cell RNA sequencing analyses indicate, and flow cytometry data confirm that B cells from circulation express transmembrane TNF, Fas ligand (FasL), lymphotoxin (LT) α1β2 and TNF-related apoptosis-inducing ligand (TRAIL). The cytotoxic activity can be inhibited by individual and, especially, combined blockade of the four transmembrane TNFSF ligands. B cells from tumor-bearing head and neck squamous cell carcinoma patients express lower levels of TNFSF ligands and are less cytotoxic than those isolated from healthy individuals. In conclusion, we demonstrate that B cells have the innate capacity to mediate anti-cancer cytotoxicity through concurrent activity of multiple plasma membrane-associated TNFSF ligands, that this mechanism is deficient in cancer patients and that it may be part of a general cancer immunosurveillance mechanism.

Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

Infection cycles of viruses are highly dependent on membrane-associated host factors. To uncover the infection cycle of Bamboo mosaic virus (BaMV) in detail, we purified the membrane-associated viral complexes from infected Nicotiana benthamiana plants and analyzed the involved host factors. Four isoforms of voltage-dependent anion channel (VDAC) proteins on the outer membrane of mitochondria were identified due to their upregulated expression in the BaMV complex-enriched membranous fraction. Results from loss- and gain-of-function experiments indicated that NbVDAC2, -3, and -4 are essential for efficient BaMV accumulation. During BaMV infection, all NbVDACs concentrated into larger aggregates, which overlapped and trafficked with BaMV virions to the structure designated as the "dynamic BaMV-induced complex." Besides the endoplasmic reticulum and mitochondria, BaMV replicase and double-stranded RNAs were also found in this complex, suggesting the dynamic BaMV-induced complex is a replication complex. Yeast two-hybrid and pull-down assays confirmed that BaMV triple gene block protein 1 (TGBp1) could interact with NbVDACs. Confocal microscopy revealed that TGBp1 is sufficient to induce NbVDAC aggregates, which suggests that TGBp1 may play a pivotal role in the NbVDAC-virion complex. Collectively, these findings indicate that NbVDACs may associate with the dynamic BaMV-induced complex via TGBp1 and NbVDAC2, -3, or -4 and can promote BaMV accumulation. This study reveals the involvement of mitochondrial proteins in a viral complex and virus infection.

Identification and Characterization of Two Transmembrane Proteins Required for Virulence of Ustilago maydis.

Smut fungi comprise a large group of biotrophic phytopathogens infecting important crops such as wheat and corn. Through the secretion of effector proteins, the fungus actively suppresses plant immune reactions and modulates its host’s metabolism. Consequently, how soluble effector proteins contribute to virulence is already characterized in a range of phytopathogens. However, membrane-associated virulence factors have been much less studied to date. Here, we investigated six transmembrane (TM) proteins that show elevated gene expression during biotrophic development of the maize pathogen Ustilago maydis. We show that two of the six proteins, named Vmp1 and Vmp2 (virulence-associated membrane protein), are essential for the full virulence of U. maydis. The deletion of the corresponding genes leads to a substantial attenuation in the virulence of U. maydis. Furthermore, both are conserved in various related smuts and contain no domains of known function. Our biochemical analysis clearly shows that Vmp1 and Vmp2 are membrane-associated proteins, potentially localizing to the U. maydis plasma membrane. Mass photometry and light scattering suggest that Vmp1 mainly occurs as a monomer, while Vmp2 is dimeric. Notably, the large and partially unstructured C-terminal domain of Vmp2 is crucial for virulence while not contributing to dimerization. Taken together, we here provide an initial characterization of two membrane proteins as virulence factors of U. maydis.

The Novel Membrane-Associated Auxiliary Factors AuxA and AuxB Modulate β-lactam Resistance in MRSA by stabilizing Lipoteichoic Acids

A major determinant of β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. However, full expression of the resistance phenotype requires auxiliary factors. We identified two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003) in a screen against mutants with increased susceptibility to β-lactams in the MRSA strain JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced β-lactam susceptibility in community-, hospital- and livestock associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid synthesis (LTA) and alanylation pathways, and released LTA even in the absence of β-lactams. The β-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, we observed enhanced survival of larvae inoculated with either auxA or auxB mutants compared to the wild type strain following treatment with amoxicillin. Collectively, our results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to β-lactams and combat MRSA infections.

Comprehensive analysis of NAC transcription factor family uncovers drought and salinity stress response in pearl millet (Pennisetum glaucum)

BackgroundPearl millet (Pennisetum glaucum) is a cereal crop that possesses the ability to withstand drought, salinity and high temperature stresses. The NAC [NAM (No Apical Meristem), ATAF1 (Arabidopsis thaliana Activation Factor 1), and CUC2 (Cup-shaped Cotyledon)] transcription factor family is one of the largest transcription factor families in plants. NAC family members are known to regulate plant growth and abiotic stress response. Currently, no reports are available on the functions of the NAC family in pearl millet.ResultsOur genome-wide analysis found 151 NAC transcription factor genes (PgNACs) in the pearl millet genome. Thirty-eight and 76 PgNACs were found to be segmental and dispersed duplicated respectively. Phylogenetic analysis divided these NAC transcription factors into 11 groups (A-K). Three PgNACs (− 073, − 29, and − 151) were found to be membrane-associated transcription factors. Seventeen other conserved motifs were found in PgNACs. Based on the similarity of PgNACs to NAC proteins in other species, the functions of PgNACs were predicted. In total, 88 microRNA target sites were predicted in 59 PgNACs. A previously performed transcriptome analysis suggests that the expression of 30 and 42 PgNACs are affected by salinity stress and drought stress, respectively. The expression of 36 randomly selected PgNACs were examined by quantitative reverse transcription-PCR. Many of these genes showed diverse salt- and drought-responsive expression patterns in roots and leaves. These results confirm that PgNACs are potentially involved in regulating abiotic stress tolerance in pearl millet.ConclusionThe pearl millet genome contains 151 NAC transcription factor genes that can be classified into 11 groups. Many of these genes are either upregulated or downregulated by either salinity or drought stress and may therefore contribute to establishing stress tolerance in pearl millet.

PSV-27 Program Chair Poster Pick: Expression QTL mapping for meat quality in beef cattle

Abstract Expression QTL mapping provides information about genetic variant with modulatory effects on gene expression which are useful for understanding the genetic architecture of complex phenotypes. This mapping allows for uncovering of genomic regions associated with transcription regulation of genes which can be related to phenotypic variation when they colocalize with QTLs (cis and trans effects), providing a molecular basis for the phenotype-genotype association. The objectives of the present research were to perform eQTL mapping for meat quality traits in longissimus dorsi muscle and to uncover genes whose expression is influenced by local or distant genetic variation. A total of 120 steers from the University of Florida Beef Unit multibreed Angus-Brahman herd born between 2013 and 2014 were used in this study. The first three principal components from a principal component analysis for all meat quality phenotypes were used to construct a meat quality index. Eighty animals were selected based on extreme meat quality index for mRNA sequencing and 100 bp paired-end reads were mapped against to the Btau_4.6.1 reference genome. eQTL mapping was performed using 112,042 SNPs and 8,588 genes. A cis QTL was defined as an SNP located no more than 1 Mb upstream of the transcription start site or downstream of the transcription end site of an annotated gene. Polymorphisms associated with expression of at least 20 genes in the case of eQTL were considered hot spots. The harboring or adjacent gene was defined as master regulators. Multiple cis eQTLs and sQTLs effects were identified and genes such as LSM2, SOAT1, TTN and TEK are a few examples of potential expression and splicing regulatory genes. A total of 27 expression and 13 splicing master regulator genes were uncovered, mainly cytoskeletal or membrane-associated proteins, transcription factors and DNA methylases.

Pancreatic Cancer Cells Require the Transcription Factor MYRF to Maintain ER Homeostasis.

Many tumors of endodermal origin are composed of highly secretory cancer cells that must adapt endoplasmic reticulum (ER) activity to enable proper folding of secreted proteins and prevent ER stress. We found that pancreatic ductal adenocarcinomas (PDACs) overexpress the myelin regulatory factor (MYRF), an ER membrane-associated transcription factor (TF) released by self-cleavage. MYRF was expressed in the well-differentiated secretory cancer cells, but not in the poorly differentiated quasi-mesenchymal cells that coexist in the same tumor. MYRF expression was controlled by the epithelial identity TF HNF1B, and it acted to fine-tune the expression of genes encoding highly glycosylated, cysteine-rich secretory proteins, thus preventing ER overload. MYRF-deficient PDAC cells showed signs of ER stress, impaired proliferation, and an inability to form spheroids invitro, while invivo they generated highly secretory but poorly proliferating and hypocellular tumors. These data indicate a role of MYRF in the control of ER homeostasis in highly secretory PDAC cells.

A membrane-associated NAC transcription factor OsNTL3 is involved in thermotolerance in rice.

SummaryHeat stress induces misfolded protein accumulation in endoplasmic reticulum (ER), which initiates the unfolded protein response (UPR) in plants. Previous work has demonstrated the important role of a rice ER membrane‐associated transcription factor OsbZIP74 (also known as OsbZIP50) in UPR. However, how OsbZIP74 and other membrane‐associated transcription factors are involved in heat stress tolerance in rice is not reported. In the current study, we discovered that OsNTL3 is required for heat stress tolerance in rice. OsNTL3 is constitutively expressed and up‐regulated by heat and ER stresses. OsNTL3 encodes a NAC transcription factor with a predicted C‐terminal transmembrane domain. GFP‐OsNTL3 relocates from plasma membrane to nucleus in response to heat stress and ER stress inducers. Loss‐of‐function mutation of OsNTL3 confers heat sensitivity while inducible expression of the truncated form of OsNTL3 without the transmembrane domain increases heat tolerance in rice seedlings. RNA‐Seq analysis revealed that OsNTL3 regulates the expression of genes involved in ER protein folding and other processes. Interestingly, OsNTL3 directly binds to OsbZIP74 promoter and regulates its expression in response to heat stress. In turn, up‐regulation of OsNTL3 by heat stress is dependent on OsbZIP74. Thus, our work reveals the important role of OsNTL3 in thermotolerance, and a regulatory circuit mediated by OsbZIP74 and OsNTL3 in communications among ER, plasma membrane and nucleus under heat stress conditions.

Preclinical translation of exosomes derived from mesenchymal stem/stromal cells.

Exosomes are nanovesicles secreted by virtually all cells. Exosomes mediate the horizontal transfer of various macromolecules previously believed to be cell‐autonomous in nature, including nonsecretory proteins, various classes of RNA, metabolites, and lipid membrane‐associated factors. Exosomes derived from mesenchymal stem/stromal cells (MSCs) appear to be particularly beneficial for enhancing recovery in various models of disease. To date, there have been more than 200 preclinical studies of exosome‐based therapies in a number of different animal models. Despite a growing number of studies reporting the therapeutic properties of MSC‐derived exosomes, their underlying mechanism of action, pharmacokinetics, and scalable manufacturing remain largely outstanding questions. Here, we review the global trends associated with preclinical development of MSC‐derived exosome‐based therapies, including immunogenicity, source of exosomes, isolation methods, biodistribution, and disease categories tested to date. Although the in vivo data assessing the therapeutic properties of MSC‐exosomes published to date are promising, several outstanding questions remain to be answered that warrant further preclinical investigation.

Plasma Membrane-Associated Restriction Factors and Their Counteraction by HIV-1 Accessory Proteins.

The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host’s attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.

A Functional Unfolded Protein Response Is Required for Normal Vegetative Development.

The unfolded protein response (UPR) is activated in plants in response to endoplasmic reticulum stress and plays an important role in mitigating stress damage. Multiple factors act in the UPR, including the membrane-associated transcription factor, BASIC LEUCINE ZIPPER 17 (bZIP17), and the membrane-associated RNA splicing factor, INOSITOL REQUIRING ENZYME1 (IRE1). We have analyzed an Arabidopsis (Arabidopsis thaliana) ire1a ire1b bzip17 triple mutant, with defects in stress signaling, and found that the mutant is also impaired in vegetative plant growth under conditions without externally applied stress. This raised the possibility that the UPR functions in plant development in the same manner as it does in responding to stress. bZIP17 is mobilized to the nucleus in response to stress, and through the analysis of a mobilization-defective bZIP17 mutant, we found that to support normal plant development bZIP17 must be capable of mobilization. Likewise, through the analysis of ire1 mutants defective in either protein kinase or RNase activities, we found that both must be operative to promote normal development. These findings demonstrate that the UPR, which is associated with stress responses in plants, also functions under unstressed conditions to support normal development.

Tail domains of myosin-1e regulate phosphatidylinositol signaling and F-actin polymerization at the ventral layer of podosomes.

During podosome formation, distinct phosphatidylinositol 3,4,5-trisphosphate lipid (PI(3,4,5)P3) production and F-actin polymerization take place at integrin-mediated adhesions. Membrane-associated actin regulation factors, such as myosin-1, serve as key molecules to link phosphatidylinositol signals to podosome assembly. Here, we report that long-tailed myosin-1e (Myo1e) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3-dependent manner. The combination of TH1 and TH2 (TH12) of Myo1e tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 KR2A (K772A and R782A) becomes dissociated from the plasma membrane. While F-actin polymerizations are initialized from the ventral layer of the podosome, TH12 precedes the recruitment of N-WASP and Arp2/3 in the initial phase of podosome formation. Overexpression of TH12, not TH12 KR2A, impedes PI(3,4,5)P3 signaling, restrains F-actin polymerization, and inhibits podosome formation. TH12 also suppresses gelatin degradation and migration speed of invadopodia-forming A375 melanoma cells. Thus, TH12 domain of Myo1e serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome.

22 - Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI)

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membranebound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both Phosphatidylserine and polyunsaturated fatty acids were required to promoteinactivation of theZPI-PZcomplexor free ZPI, indicating that binding of thePZ-complexedor freeZPIto peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.