Anam Qudrat

Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells

The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca2+). By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca2+-activated processes.

Engineering mammalian cells to seek senescence-associated secretory phenotypes

ABSTRACT Since the removal of senescent cells in model organisms has been linked to rejuvenation and increased lifespan, senotherapies have emerged to target senescent cells for death. In particular, interleukin-6 (IL6) is a prominent senescence-associated secretory phenotype (SASP) and, thus, seeking IL6 could potentially localize engineered cells to senescent cells for therapeutic intervention. Here, we engineered a chimeric IL6 receptor (IL6Rchi) that generates a Ca2+ signal in response to IL6 stimulation. When IL6Rchi was co-expressed with an engineered Ca2+-activated RhoA (CaRQ), it enabled directed migration to IL6 in cells that have no such natural ability. Next, the removal of target cells was accomplished by the mechanism of membrane fusion and subsequent death. This work represents a first step towards engineering a cell to target senescent cells that secrete high levels of IL6. For increased specificity to senescent cells, it will likely be necessary for an engineered cell to recognize multiple SASPs simultaneously. Summary: IL6-seeking cells were engineered to target synthetic IL6 sources using a chimeric receptor and a previously engineered Ca2+-activated RhoA. Such cells could potentially be used to target senescence.

Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites

Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca2+-activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment.

Autonomous Cell Migration to CSF1 Sources via a Synthetic Protein-Based System

Inflammatory lesions, often seen in diseases such as rheumatoid arthritis, atherosclerosis and cancer, feature an acidic (i.e., low pH) microenvironment rampant with cytokines, such as CSF1. For potential therapeutic intervention targeted at these CSF1 sources, we have assembled a system of four proteins inside a cell (i.e., HEK293) that initially had no natural CSF1-seeking ability. This system included a newly engineered CSF1 chimera receptor (named CSF1Rchi), the previously engineered Ca2+ activated RhoA (i.e., CaRQ), vesicular stomatitis virus glycoprotein G (VSVG) and thymidine kinase (TK). The binding of CSF1 to the CSF1Rchi generated a Ca2+ signal that activated CaRQ-mediated cellular blebbing, allowing autonomous cell migration toward the CSF1 source. Next, the VSVG protein allowed these engineered cells to fuse with the CSF1 source cells, upon low pH induction. Finally, these cells underwent death postganciclovir treatment, via the TK suicide mechanism. Hence, this protein system could potentially serve as the basis of engineering a cell to target inflammatory lesions in diseases featuring a microenvironment with high levels of CSF1 and low pH.

The spatiotemporal relationship between local Ca2+ signaling and P2X2R-activated membrane blebbing

Mammalian P2X receptors (P2XRs), a family of seven ionotropic purinergic receptors, function as ion channels modulating diverse cellular processes such as secretion, apoptosis and proliferation in response to extracellular ATP. Previously, it was shown that upon ATP stimulus, the P2X7 receptor (a member of P2XR family) triggers plasma membrane (PM) blebbing in HEK293 cells. In this study, we demonstrate that this phenomenon extends to another member of the P2XR family-P2X2 receptor (P2X2R). Similar to P2X7 receptor, P2X2R blebbing is dependent on Ca(2+)-calmodulin and ROCK-I. To elucidate the spatiotemporal relationship between Ca(2+) signaling and blebbing, protein biosensors and switches were used to image and generate Ca(2+) signals, respectively, while observing PM blebbing in cells. Blebbing cannot be initiated by Ca(2+) influx from the endoplasmic reticulum or by Ca(2+) transport across the PM by other Ca(2+) channels. To trigger blebbing, it is necessary for Ca(2+) to enter specifically through the P2X2R. Lastly, a local Ca(2+) signal near a fragment that encodes the intracellular P2X2R C-terminus tail is sufficient to trigger blebbing.

Engineered cell migration to lesions linked to autoimmune disease

The damaging and degenerative effects in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and Crohns disease often manifests as the formation of lesions that feature a high local concentration of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF along with other pro‐inflammatory factors form a positive feedback loop that ultimately perpetuate the lesions. Hence, to engineer chemotaxis to GM‐CSF, we created a new chimeric GM‐CSF receptor alpha subunit (GMRchi) that was coupled with a previously engineered Ca2+‐activated RhoA. When these proteins were expressed in mammalian cells, it allowed migration to chemical and cellular sources of GM‐CSF. As a possible therapeutic intervention, we further implemented the mechanism of cell–cell membrane fusion and subsequent death. Since the microenvironment of lesions is more than just GM‐CSF secretion, the further ability to recognize a combination of other features such as tissue markers will be needed for greater specificity. Nonetheless, this work represents a first step to enable cell‐based therapy of autoimmune lesions.

Antibody-Based Fusion Proteins Allow Ca2+ Rewiring to Most Extracellular Ligands

The Ca2+ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca2+ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca2+ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca2+ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca2+ signal upon oligomerization. Using protein fusions between antibody variants (i.e., nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca2+ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumor necrosis factor alpha and soluble CD14. The Ca2+ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state, but instead only required an increase in the oligomeric state. The Ca2+ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca2+ indicator or with an engineered Ca2+ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively.

LOV2-Controlled Photoactivation of Protein Trans -Splicing

Protein trans-splicing is a posttranslational modification that joins two protein fragments together via a peptide a bond in a process that does not require exogenous cofactors. Towards achieving cellular control, synthetically engineered systems have used a variety of stimuli such as small molecules and light. Recently, split inteins have been engineered to be photoactive by the LOV2 domain (named LOVInC). Herein, we discuss (1) designing of LOV2-activated target proteins (e.g., inteins), (2) selecting feasible splice sites for the extein, and (3) imaging cells that express LOVInC-based target exteins.

Receptor-Localized Ca2+ Signaling Activates P2X2 Receptor Changing Cytoskeletal Morphology

Mammalian P2X receptors (P2XRs), a family of seven ionotropic purinergic receptors, function as cationion-selective (i.e. Ca2+, K+, Na+, Cl-) channels modulating diverse cellular processes such as secretion, apoptosis and proliferation in response to extracellular ATP. Previously, it was shown that upon ATP stimulus, the P2X7 receptor (a member of P2XRs superfamily) triggers plasma membrane (PM) blebbing in HEK293 cells. In this study, we further demonstrate that this phenomenon extends to another member of the P2XRs superfamily - P2X2 receptor (P2X2R). Similar to P2X7 receptor, P2X2R blebbing is dependent on Ca2+- calmodulin and ROCK-I. To elucidate the spatiotemporal relationship between Ca2+ signaling and blebbing, protein biosensors and switches were used to image and generate Ca2+ signals, respectively, while observing PM blebbing in cells. Blebbing cannot be initiated by Ca2+ influx from the endoplasmic reticulum or by Ca2+ transport across the PM by other Ca2+ channels. To trigger blebbing, it is necessary for Ca2+ to enter through the P2X2R. Lastly, a local Ca2+ signal near a fragment that encodes the intracellular P2X2R C-terminus tail is sufficient to trigger blebbing.

Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells.

BackgroundTo achieve synthetic control over how a cell responds to other cells or the extracellular environment, it is important to reliably engineer proteins that can traffic and span the plasma membrane. Using a modular approach to assemble proteins, we identified the minimum necessary components required to engineer such membrane-spanning proteins with predictable orientation in mammalian cells.ResultsWhile a transmembrane domain (TM) fused to the N-terminus of a protein is sufficient to traffic it to the endoplasmic reticulum (ER), an additional signal peptidase cleavage site downstream of this TM enhanced sorting out of the ER. Next, a second TM in the synthetic protein helped anchor and accumulate the membrane-spanning protein on the plasma membrane. The orientation of the components of the synthetic protein were determined through measuring intracellular Ca2+ signaling using the R-GECO biosensor and through measuring extracellular quenching of yellow fluorescent protein variants by saturating acidic and salt conditions.ConclusionsThis work forms the basis of engineering novel proteins that span the plasma membrane to potentially control intracellular responses to extracellular conditions.