Reconstructing Signaling Networks Using Biosensor Barcoding.

Understanding how signaling networks are regulated offers valuable insights into how cells and organisms react to internal and external stimuli and is crucial for developing novel strategies to treat diseases. To achieve this, it is necessary to delineate the intricate interactions between the nodes in the network, which can be accomplished by measuring the activities of individual nodes under perturbation conditions. To facilitate this, we have recently developed a biosensor barcoding technique that enables massively multiplexed tracking of numerous signaling activities in live cells using genetically encoded fluorescent biosensors. In this chapter, we detail how we employed this method to reconstruct the EGFR signaling network by systematically monitoring the activities of individual nodes under perturbations.

Arginine-linked HPV-associated E7 displaying bacteria-derived outer membrane vesicles as a potent antigen-specific cancer vaccine.

BACKGROUND: Bacteria-based cancer therapy have demonstrated innovative strategies to combat tumors. Recent studies have focused on gram-negative bacterial outer membrane vesicles (OMVs) as a novel cancer immunotherapy strategy due to its intrinsic properties as a versatile carrier. METHOD: Here, we developed an Human Papillomavirus (HPV)-associated E7 antigen displaying Salmonella-derived OMV vaccine, utilizing a Poly(L-arginine) cell penetrating peptide (CPP) to enhance HPV16 E7 (aa49-67) H-2 Db and OMV affinity, termed SOMV-9RE7. RESULTS: Due to OMV's intrinsic immunogenic properties, SOMV-9RE7 effectively activates adaptive immunity through antigen-presenting cell uptake and antigen cross-presentation. Vaccination of engineered OMVs shows immediate tumor suppression and recruitment of infiltrating tumor-reactive immune cells. CONCLUSION: The simplicity of the arginine coating strategy boasts the versatility of immuno-stimulating OMVs that can be broadly implemented to personalized bacterial immunotherapeutic applications.

Salmonella immunotherapy engineered with highly efficient tumor antigen coating establishes antigen-specific CD8+ T cell immunity and increases in antitumor efficacy with type I interferon combination therapy.

Bacteria-based cancer therapy employs various strategies to combat tumors, one of which is delivering tumor-associated antigen (TAA) to generate specific immunity. Here, we utilized a poly-arginine extended HPV E7 antigen (9RE7) for attachment on Salmonella SL7207 outer membrane to synthesize the bacterial vaccine Salmonella-9RE7 (Sal-9RE7), which yielded a significant improvement in the amount of antigen presentation compared to the previous lysine-extended antigen coating strategy. In TC-1 tumor mouse models, Sal-9RE7 monotherapy decreased tumor growth by inducing E7 antigen-specific immunity. In addition, pairing Sal-9RE7 with adjuvant Albumin-IFNß (Alb-IFNß), a protein cytokine fusion, the combination significantly increased the antitumor efficacy and enhanced immunogenicity in the tumor microenvironment (TME). Our study made a significant contribution to personalized bacterial immunotherapy via TAA delivery and demonstrated the advantage of combination therapy.

Targeting Ras signaling excitability in cancer cells through combined inhibition of FAK and PI3K.

The Ras/PI3K/ERK signaling network is frequently mutated in various human cancers including cervical cancer and pancreatic cancer. Previous studies showed that the Ras/PI3K/ERK signaling network displays features of excitable systems including propagation of activity waves, all-or-none responses, and refractoriness. Oncogenic mutations lead to enhanced excitability of the network. A positive feedback loop between Ras, PI3K, the cytoskeleton, and FAK was identified as a driver of excitability. In this study, we investigated the effectiveness of targeting signaling excitability by inhibiting both FAK and PI3K in cervical and pancreatic cancer cells. We found that the combination of FAK and PI3K inhibitors synergistically suppressed the growth of select cervical and pancreatic cancer cell lines through increased apoptosis and decreased mitosis. In particular, FAK inhibition caused downregulation of PI3K and ERK signaling in cervical cancer but not pancreatic cancer cells. Interestingly, PI3K inhibitors activated multiple receptor tyrosine kinases (RTKs), including insulin receptor and IGF-1R in cervical cancer cells, as well as EGFR, Her2, Her3, Axl, and EphA2 in pancreatic cancer cells. Our results highlight the potential of combining FAK and PI3K inhibition for treating cervical and pancreatic cancer, although appropriate biomarkers for drug sensitivity are needed, and concurrent targeting of RTKs may be required for resistant cells.

Imaging and analysis for simultaneous tracking of fluorescent biosensors in barcoded cells.

We recently developed a biosensor barcoding approach for highly multiplexed tracking of molecular activities in live cells. In this protocol, we detail the labeling of cells expressing different genetically encoded fluorescent biosensors with a pair of barcoding proteins and parallel imaging. Signals from cells with the same barcodes are then pooled together to obtain the dynamics of the corresponding biosensor activity. We describe the steps involved in cell barcoding, image acquisition, and analysis by deep learning models. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).

Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

Publisher Correction: Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

The original version of this Article contained an error in the spelling of the author Jr-Ming Yang, which was incorrectly given as J.-Ming Yang. This has now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

In the original version of this Article, the label "RTK" in Figure 6a was inadvertently changed to "RTE". This has now been corrected in the PDF and HTML versions of the Article.

Integrating chemical and mechanical signals through dynamic coupling between cellular protrusions and pulsed ERK activation.

The Ras-ERK signaling pathway regulates diverse cellular processes in response to environmental stimuli and contains important therapeutic targets for cancer. Recent single cell studies revealed stochastic pulses of ERK activation, the frequency of which determines functional outcomes such as cell proliferation. Here we show that ERK pulses are initiated by localized protrusive activities. Chemically and optogenetically induced protrusions trigger ERK activation through various entry points into the feedback loop involving Ras, PI3K, the cytoskeleton, and cellular adhesion. The excitability of the protrusive signaling network drives stochastic ERK activation in unstimulated cells and oscillations upon growth factor stimulation. Importantly, protrusions allow cells to sense combined signals from substrate stiffness and the growth factor. Thus, by uncovering the basis of ERK pulse generation we demonstrate how signals involved in cell growth and differentiation are regulated by dynamic protrusions that integrate chemical and mechanical inputs from the environment.

Opening the conformation is a master switch for the dual localization and phosphatase activity of PTEN.

Tumor suppressor PTEN mainly functions at two subcellular locations, the plasma membrane and the nucleus. At the plasma membrane, PTEN dephosphorylates the tumorigenic second messenger PIP3, which drives cell proliferation and migration. In the nucleus, PTEN controls DNA repair and genome stability independently of PIP3. Whereas the concept that a conformational change regulates protein function through post-translational modifications has been well established in biology, it is unknown whether a conformational change simultaneously controls dual subcellular localizations of proteins. Here, we discovered that opening the conformation of PTEN is the crucial upstream event that determines its key dual localizations of this crucial tumor suppressor. We identify a critical conformational switch that regulates PTEN's localization. Most PTEN molecules are held in the cytosol in a closed conformation by intramolecular interactions between the C-terminal tail and core region. Dephosphorylation of the tail opens the conformation and exposes the membrane-binding regulatory interface in the core region, recruiting PTEN to the membrane. Moreover, a lysine at residue 13 is also exposed and when ubiquitinated, transports PTEN to the nucleus. Thus, opening the conformation of PTEN is a key mechanism that enhances its dual localization and enzymatic activity, providing a potential therapeutic strategy in cancer treatments.

Engineering PTEN function: membrane association and activity.

Many tumors are associated with deficiency of the tumor suppressor, PTEN, a PIP3 phosphatase that turns off PIP3 signaling. The major site of PTEN action is the plasma membrane, where PIP3 is produced by PI3 kinases. However, the mechanism and functional importance of PTEN membrane recruitment are poorly defined. Using the heterologous expression system in which human PTEN is expressed in Dictyostelium discoideum, we defined the molecular mechanisms that regulate the membrane-binding site through inhibitory interactions with the phosphorylated C-terminal tail. In addition, we potentiated mechanisms that mediate PTEN membrane association and engineered an enhanced PTEN with increased tumor suppressor functions. Moreover, we identified a new class of cancer-associated PTEN mutations that are specifically defective in membrane association. In this review, we summarize recent advances in PTEN-membrane interactions and methods useful in addressing PTEN function.

Engineering ePTEN, an enhanced PTEN with increased tumor suppressor activities.

The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a key regulator of cell proliferation, survival, and migration and the enzyme that dephosphorylates it, phosphatase and tensin homolog (PTEN), is an important tumor suppressor. As excess PIP3 signaling is a hallmark of many cancers, its suppression through activation of PTEN is a potential cancer intervention. Using a heterologous expression system in which human PTEN-GFP is expressed in Dictyostelium cells, we identified mutations in the membrane-binding regulatory interface that increase the recruitment of PTEN to the plasma membrane due to enhanced association with PI(4,5)P2. We engineered these into an enhanced PTEN (ePTEN) with approximately eightfold increased ability to suppress PIP3 signaling. Upon expression in human cells, ePTEN decreases PIP3 levels in the plasma membrane; phosphorylation of AKT, a major downstream event in PIP3 signaling; and cell proliferation and migration. Thus, the activation of PTEN can readjust PIP3 signaling and may serve as a feasible target for anticancer therapies.

The cis-acting signals that target proteins to exosomes and microvesicles.

Proteins bud from cells in small single-membraned vesicles (~50-250 nm) that have the same topology as the cell. Known variously as exosomes and microvesicles (EMVs), these extracellular organelles are enriched for specific proteins, lipids, carbohydrates and nucleic acids. EMV biogenesis plays critical roles in protein quality control and cell polarity, and, once released, EMVs can transmit signals and molecules to neighbouring cells via a non-viral pathway of intercellular vesicle traffic. In the present paper, we discuss the cis-acting targeting signals that target proteins to EMVs and mediate protein budding from the cell.

Protein targeting to exosomes/microvesicles by plasma membrane anchors.

Animal cells secrete small vesicles, otherwise known as exosomes and microvesicles (EMVs). A short, N-terminal acylation tag can target a highly oligomeric cytoplasmic protein, TyA, into secreted vesicles (Fang, Y., Wu, N., Gan, X., Yan, W., Morell, J. C., and Gould, S. J. (2007) PLoS Biol. 5, 1267-1283). However, it is not clear whether this is true for other membrane anchors or other highly oligomeric, cytoplasmic proteins. We show here that a variety of plasma membrane anchors can target TyA-GFP to sites of vesicle budding and into EMVs, including: (i) a myristoylation tag; (ii) a phosphatidylinositol-(4,5)-bisphosphate (PIP(2))-binding domain; (iii), a phosphatidylinositol-(3,4,5)-trisphosphate-binding domain; (iv) a prenylation/palmitoylation tag, and (v) a type-1 plasma membrane protein, CD43. However, the relative budding efficiency induced by these plasma membrane anchors varied over a 10-fold range, from 100% of control (AcylTyA-GFP) for the myristoylation tag and PIP(2)-binding domain, to one-third or less for the others, respectively. Targeting TyA-GFP to endosome membranes by fusion to a phosphatidylinositol 3-phosphate-binding domain induced only a slight budding of TyA-GFP, ∼2% of control, and no budding was observed when TyA-GFP was targeted to Golgi membranes via a phosphatidylinositol 4-phosphate-binding domain. We also found that a plasma membrane anchor can target two other highly oligomeric, cytoplasmic proteins to EMVs. These observations support the hypothesis that plasma membrane anchors can target highly oligomeric, cytoplasmic proteins to EMVs. Our data also provide additional parallels between EMV biogenesis and retrovirus budding, as the anchors that induced the greatest budding of TyA-GFP are the same as those that mediate retrovirus budding.

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane.

Exosomes are secreted, single membrane organelles of approximately 100 nm diameter. Their biogenesis is typically thought to occur in a two-step process involving (1) outward vesicle budding at limiting membranes of endosomes (outward = away from the cytoplasm), which generates intralumenal vesicles, followed by (2) endosome-plasma membrane fusion, which releases these internal vesicles into the extracellular milieu as exosomes. In this study, we present evidence that certain cells, including Jurkat T cells, possess discrete domains of plasma membrane that are enriched for exosomal and endosomal proteins, retain the endosomal property of outward vesicle budding, and serve as sites of immediate exosome biogenesis. It has been hypothesized that retroviruses utilize the exosome biogenesis pathway for the formation of infectious particles. In support of this, we find that Jurkat T cells direct the key budding factor of HIV, HIV Gag, to these endosome-like domains of plasma membrane and secrete HIV Gag from the cell in exosomes.

Engineering a thermostable protein with two DNA-binding domains using the hyperthermophile protein Sac7d.

The acid- and thermostable Sac7d is a small, non-specific DNA-binding protein of the hyperthermophile archaea Sulfolobus acidocaldarius. In this study, Sac7d was employed as a structural unit in the design of a thermostable protein containing two putative DNA-binding domains. By linking two Sac7d proteins together and comparing the DNA interaction of dimer to that of monomer, this study may provide structural insights into other dimeric DNA-binding proteins. The engineered protein, Sac7dK66C, was over-expressed and purified. Dimeric Sac7d was obtained by cross-linking two mutant Sac7d molecules through the C-terminal disulfide bond. Thermal stability and DNA-binding ability of dimeric Sac7d were assessed and compared to those of wild type Sac7d by gel retardation assay, circular dichroism spectroscopy, and crystallization experiments. Dimeric Sac7d was shown to be equally thermostable as wild type, and its ability to stabilize DNA duplex is the same as wild type. However, the interaction of dimeric Sac7d with DNA diverged from that of wild type, suggesting different DNA-binding modes for dimeric Sac7d. In addition, a large difference in extinction coefficient was observed in all dimer/DNA CD spectra, which was reminiscent of the spectrum of Psi-DNA. Conjugation of various chemical groups to mutant Sac7d is possible through the C-terminal thiol group. This offers a possible approach in the design of a thermostable biomolecule with novel functions.