A C-terminal peptide of TFPI-1 facilitates cytosolic delivery of nucleic acid cargo into mammalian cells.

Efficient intracellular nucleic acid delivery into mammalian cells remains a long-standing challenge owing to poor cell permeability and uptake of naked nucleic acids across the cell membrane and limited cargo stability. Conventional delivery methods have several drawbacks, such as cytotoxicity, limited cell-type applicability, low efficiency, hindrances that limit the potential of oligonucleotide delivery in functional genomics, therapeutics and diverse research applications. Thus, new approaches that are robust, safe, effective and valid across multiple cell types are much needed. Here, we demonstrate that GGL27, a TFPI-1-derived novel cationic host defence peptide, facilitates the delivery of nucleic acid cargo into the cytosol of a range of mammalian cells. The GGL27 peptide is non-cytotoxic and is internalized in a broad range of mammalian cell-types, including transformed cell lines and primary cells. GGL27 spontaneously forms complexes with nucleic acids of variable sizes, protects them from nuclease degradation, and delivers cargo effectively. Together, our observations demonstrate the versatile cell-penetrating property of GGL27, providing an excellent template for developing a simple, non-toxic peptide-based cytosolic delivery tool for wide use in biomedical research.

CG-NAP/Kinase Interactions Fine-Tune T Cell Functions.

CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions. As a result, new binding partners of CG-NAP are continually being uncovered. Here, we review recent advances in CG-NAP research, focusing on its interactions with kinases in T cells with an emphasis on the possible role of this anchoring protein as a target for therapeutic intervention in immune-mediated diseases.

Isolation of Human Peripheral Blood T-Lymphocytes.

Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays.

A Laboratory Model to Study T-Cell Motility.

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLß2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.

GapmeR-Mediated Gene Silencing in Motile T-Cells.

Gene silencing is an important method to study gene functions in health and diseases. While there are various techniques that are applied to knockdown specific gene(s) of interest, they have certain limitations in application to T-lymphocytes. T-cells are "hard-to-transfect" cells and are recalcitrant to transfection reagents. Here, we describe the use of novel cell-permeating antisense molecules, called "GapmeR", to knockdown specific gene(s) in human primary T-cells.

Immunometabolomic Phenotyping of Motile T-Cells.

The immune system and its components defend our body against diverse pathogens and help in maintaining tissue homeostasis. Immune cells are highly dynamic in terms of their growth, migration, differentiation, and effector functions, and adopt diverse metabolic configurations to respond to varying immunological challenges. Growing body of evidence suggests that metabolic pathways fuel immune cells for their functioning, including T-cell migration to the site of infection. This chapter provides detailed methodology for the efficient extraction of T-cell metabolites for successful downstream immunometabolomic profiling of motile T-lymphocytes.

Enzyme-Linked Immunosorbent Assay for T-Cell Dependent Immunogenicity Assessment of Therapeutic Peptides.

Immunogenicity assessment of therapeutic peptides, proteins, oligonucleotides, and hybrid molecules, such as nucleopeptides, is a major aspect in understanding their safety and efficacy. Both T-cell independent and dependent immune reactions contribute to an immunogenic response against antigen, including secretion of cytokines and production of an antigen-specific antibody. Various assays exist for detecting and quantifying such immunogenic responses by human T-cells ex vivo or in mouse serum, which primarily include enzyme-linked immunosorbent assay (ELISA, direct and indirect), flow-cytometry and surface plasmon resonance (SPR). ELISA is a popular choice due to its robustness, reliability, sensitivity, ease of automation, and the requirement of simple equipment commonly available in most molecular biology and biochemistry laboratories. The chapter describes the detailed protocol of cytokine analysis by an ELISA method and highlights few crucial steps to be considered while performing the assay for successful immunogenicity studies.

Combating Microbial Contamination with Robust Polymeric Nanofibers: Elemental Effect on the Mussel-Inspired Cross-Linking of Electrospun Gelatin.

Designing biocompatible nanofibrous mats capable of preventing microbial colonization from resident and nosocomial bacteria for an extended period remains an unmet clinical need. In the present work, we designed antibiotic free durable antimicrobial nanofiber mats by taking advantage of synergistic interactions between polydopamine (pDA) and metal ions with varying degree of antimicrobial properties (Ag+, Mg2+, Ca2+, and Zn2+). Microscopic analysis showed successful pDA-mediated cross-linking of the gelatin nanofibers, which further improved by the inclusion of Ag+, Mg2+, and Ca2+ ions as supported by mechanical and thermal studies. Spectroscopic results reinforce the presence of strong interactions between pDA and metal ions in the composite nanofibers, leading to generation of robust polymeric nanofibers. We further showed that strong pDA-Ag interactions attenuated the cell cytotoxicity and anticell proliferative properties of silver ions for immortalized keratinocytes and primary human dermal fibroblasts. pDA-Ca2+/Zn2+ interactions rendered the composite structure sterile against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains, whereas the silver ion-incorporated composite mats displayed broad spectrum antibacterial activity against both Gram-positive/-negative bacteria and yeast strains. We showed that the strong pDA-Ag interactions help retaining long-term antimicrobial activity of the mats for at least 40 days while attenuating mammalian cell cytotoxicity of silver ions for skin cells. Overall, the results suggest the potential of pDA-metal ion interactions for engineering sterile nanofibrous mats and expanding the antibiotic armamentarium against drug-resistant pathogens.

Surface characteristics and antimicrobial properties of modified catheter surfaces by polypyrogallol and metal ions.

Catheter associated infections (CAIs) are the major cause of nosocomial infections leading to increased morbidity, mortality rates and economical loss. Though the antibiotic coated surface modified catheters are reported to be effective in preventing CAIs, presence of sub-lethal concentrations of antibiotics in long term instilled catheters poses a risk of development and spread of drug resistant microbial strains. Herein, we have developed an antibiotic-free alternative strategy to coat catheter surfaces using pyrogallol (PG) and metal ions (Ag+/Mg2+). Surface characteristics, antimicrobial and anti-biofilm properties with hemocompatibility of the coated catheters were studied. Structural characteristics of coated catheters were similar to the uncoated catheters with improved wettability. All the coated catheters with PG and different PG/metal ion combinations exhibited broad spectrum antibacterial activity. Catheters coated with PG/metal ions combination showed effective antibiofilm properties against MRSA strains. None of the coated catheters showed any significant hemolysis for rabbit erythrocytes. In addition, polypyrogallol (pPG) coating attenuated the hemolytic properties of silver without altering the antimicrobial properties. The inherent antimicrobial properties of the coating agent along with antimicrobial metal ions broaden the application landscape which includes coating of other medical devices, clean room construction and development of antimicrobial surfaces. The chemical formulation can also be used to design antiseptic solutions to prevent healthcare associated infections.

Centrosome- and Golgi-Localized Protein Kinase N-Associated Protein Serves As a Docking Platform for Protein Kinase A Signaling and Microtubule Nucleation in Migrating T-Cells.

Centrosome- and Golgi-localized protein kinase N-associated protein (CG-NAP), also known as AKAP450, is a cytosolic scaffolding protein involved in the targeted positioning of multiple signaling molecules, which are critical for cellular functioning. Here, we show that CG-NAP is predominantly expressed in human primary T-lymphocytes, localizes in close proximity (<0.2 µm) with centrosomal and Golgi structures and serves as a docking platform for Protein Kinase A (PKA). GapmeR-mediated knockdown of CG-NAP inhibits LFA-1-induced T-cell migration and impairs T-cell chemotaxis toward the chemokine SDF-1α. Depletion of CG-NAP dislocates PKARIIα, disrupts centrosomal and non-centrosomal microtubule nucleation, causes Golgi fragmentation, and impedes α-tubulin tyrosination and acetylation, which are important for microtubule dynamics and stability in migrating T-cells. Furthermore, we show that CG-NAP coordinates PKA-mediated phosphorylation of pericentrin and dynein in T-cells. Overall, our findings provide critical insights into the roles of CG-NAP in regulating cytoskeletal architecture and T-cell migration.

GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells.

Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to "hard-to-transfect" primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called "GapmeR", is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics.

E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy.

The protein optineurin coded by OPTN gene is involved in several functions including regulation of endocytic trafficking, autophagy and signal transduction. Certain missense mutations in the gene OPTN cause normal tension glaucoma. A glaucoma-causing mutant of optineurin, E50K, induces death selectively in retinal cells. This mutant induces defective endocytic recycling of transferrin receptor by causing inactivation of Rab8 mediated by the GTPase-activating protein, TBC1D17. Here, we have explored the mechanism of E50K-induced cell death. E50K-OPTN-induced cell death was inhibited by co-expression of a catalytically inactive mutant of TBC1D17 and also by shRNA mediated knockdown of TBC1D17. Endogenous TBC1D17 colocalized with E50K-OPTN in vesicular structures. Co-expression of transferrin receptor partially protected against E50K-induced cell death. Overexpression of the E50K-OPTN but not WT-OPTN inhibited autophagy flux. Treatment of cells with rapamycin, an inducer of autophagy, reduced E50K-OPTN-induced cell death. An LC3-binding-defective mutant of E50K-OPTN showed reduced cell death, further suggesting the involvement of autophagy. TBC1D17 localized to autophagosomes and inhibited autophagy flux dependent on its catalytic activity. Knockdown of TBC1D17 rescued cells from E50K-mediated inhibition of autophagy flux. Overall, our results suggest that E50K mutant induced death of retinal cells involves impaired autophagy as well as impaired transferrin receptor function. TBC1D17, a GTPase-activating protein for Rab GTPases, plays a crucial role in E50K-induced impaired autophagy and cell death.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

Optineurin mediates a negative regulation of Rab8 by the GTPase-activating protein TBC1D17.

Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).