Biology subjects

Syahirah, R.

Publications and source records attributed to Syahirah, R..

6 recordsLinked to original sources

bioRxiv2025

RABEP1 amplifies front signaling in neutrophil migration

Neutrophils are the first responders of our innate immune system, crucial for defense against various infections. The intricate regulation of neutrophil migration is essential for neutrophil function. However, a complete mechanistic understanding is missing. We performed a miRNA overexpression screen and identified miR-190 as a potent suppressor of neutrophil migration in zebrafish. Through a second round of small-scale screening using neutrophil-specific knockouts of putative miR-190 targets, we identified that rabep1 (Rabaptin-5, RAB GTPase binding effector protein 1) is essential for neutrophil motility and chemotaxis in zebrafish. Re-expressing full-length RABEP1 in the knockout, but not a truncated form lacking the Rab4/Rab5 binding domain, rescued cell motility. Knocking down RABEP1 in human dHL-60 cells consistently reduced cell motility. RABEP1-deficient dHL-60 cells accumulate excessive members inside the cells. The Rab5 GTP level is unaffected, but the RABEP1 knockdown cells displayed reduced PAK phosphorylation and overall reduced actin polymerization, but still appropriately polarized upon chemokine stimulation. Overexpression of dominant-negative Rab4 or Rab5 similarly inhibits neutrophil migration. Our data suggests that RABEP1 drives endosomal recycling, Rac activation, and leading-edge actin polymerization, providing significant insights into the role of the endocytic pathway in neutrophil motility.

cell biology↗

bioRxiv2023

Molecular basis for Gβγ-mediated activation ofphosphoinositide 3-kinase γ

The conversion of PIP2 to PIP3 by phosphoinositide 3-kinase {gamma} (PI3K{gamma}) is a critical step in neutrophil chemotaxis and is essential for metastasis in many types of cancer. PI3K{gamma} is activated via directed interaction with G{beta}{gamma} heterodimers released from cell-surface G protein-coupled receptors (GPCRs) responding to extracellular signals. To resolve how G{beta}{gamma} activates PI3K{gamma}, we determined cryo-EM reconstructions of PI3K{gamma}-G{beta}{gamma} complexes in the presence of various substrates/analogs, revealing two distinct G{beta}{gamma} binding sites, one on the p110{gamma} helical domain and one on the C-terminal domain of the p101 subunit. Comparison of these complexes with structures of PI3K{gamma} alone demonstrates conformational changes in the kinase domain upon G{beta}{gamma} binding similar to those induced by Ras{middle dot}GTP. Assays of variants perturbing the two G{beta}{gamma} binding sites and interdomain contacts that change upon G{beta}{gamma} binding suggest that G{beta}{gamma} not only recruits the enzyme to membranes but also allosterically controls activity via both sites. Studies in a zebrafish model examining neutrophil migration are consistent with these results. These findings set the stage for future detailed investigation of G{beta}{gamma}-mediated activation mechanisms in this enzyme family and will aid in developing drugs selective for PI3K{gamma}.

biophysics↗

bioRxiv2022

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy

Neutrophils, the most abundant white blood cells in the circulation, are closely related to cancer development and progression. Primary neutrophils from healthy donors present potent cytotoxicity against different human cancer cell lines through direct contact and via the generation of reactive oxygen species (ROS). However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with widely used chimeric antigen receptors (CARs) to enhance their anti-tumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells (hPSCs) with different synthetic CARs and successfully differentiated them into functional neutrophils by implementing a novel chemically-defined differentiation platform. Neutrophils expressing the chlorotoxin (CLTX)-T-CAR presented specific cytotoxicity against glioblastoma (GBM) cells both in monolayer and 3D cultures. In a GBM xenograft mouse model, systematically-administered CLTX-T-CAR neutrophils also displayed enhanced anti-tumor activity and prolonged animal survival compared with peripheral blood-neutrophils, hPSC-neutrophils and CLTX-NK-CAR natural killer (NK) cells. Collectively, we established a new platform for production of CAR-neutrophils, paving the way to myeloid cell-based therapeutic strategies that would complement and boost current cancer treatment approaches.

bioengineering↗

bioRxiv2021

RORA regulates neutrophil migration and activation in zebrafish

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting ROR, but not the closely related ROR{gamma}, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Ror or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, ROR regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR- 99 and ROR in regulating neutrophil migration and anti-microbial defense.

cell biology↗

bioRxiv2020

A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

Tissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

genetics↗

bioRxiv2019

Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

Neutrophil migration is essential for battling against infections but also drives chronic inflammation. Since primary neutrophils are terminally differentiated and not genetically tractable, leukemia cells such as HL-60 are differentiated into neutrophil-like cells to study mechanisms underlying neutrophil migration. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Here we apply the tet-on system to induce the expression of zebrafish miR-722 in differentiated HL-60. Overexpression of miR-722 reduced the mRNA level of genes in the chemotaxis and inflammation pathways, including RAC2. Consistently, cell migration is significantly inhibited upon induced miR-722 overexpression. Together, miR-722 is an evolutionarily conserved suppressor for neutrophil migration and signaling in zebrafish and human.

immunology↗