Biology subjects

Rayatpisheh, S.

Publications and source records attributed to Rayatpisheh, S..

4 recordsLinked to original sources

bioRxiv2022

Drug interaction mapping with proximity dependent enzyme recruiting chimeras

Proximity dependent labeling using engineered enzymes has been used extensively to identify protein-protein interactions, and map protein complexes in-vitro and in-vivo. Here, we extend the use of engineered promiscuous biotin ligases to the identification of small molecule protein targets. Chimeric bi-functional chemical probes ("recruiters") are used to effectively recruit tagged biotin ligases for proximity dependent labeling of target and target interactors. The broad applicability of this approach is demonstrated with probes developed from a multi-kinase inhibitor, a bromodomain targeting moiety, and an FKBP targeting molecule. While complementary to traditional chemo-proteomic strategies such as photo-affinity labeling (PAL), and activity-based protein profiling (ABPP), this approach is a useful addition to the target ID toolbox with opportunities for tunability based on the inherent labeling efficiencies of different engineered enzymes and control over the enzyme cellular localization.

molecular biology↗

bioRxiv2020

APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei

Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 "proximity proteome" showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily purified. IMPORTANCESleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and has allowed identification of proximity proteomes for different subdomains. This capacity opens the possibility to study the composition and function of other compartments. We further expect that this approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.

microbiology↗

bioRxiv2019

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between parasite and host compartments. The PVM is the site of several essential transport activities but the basis for organization of this membrane system is unknown. We utilized the second-generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of the PVM, identifying known and novel candidate PVM proteins. Among the best represented hits were members of a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated the function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by profound changes in vacuole ultrastructure, including increased separation of the PVM and PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant version of EXP1 lacking a residue important for this activity in vitro still provides substantial rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM organization.\n\nImportanceLike other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. Although the vacuole is the site of several transport activities essential to parasite survival, little is known about its organization. To explore vacuole biology, we adopted recently developed proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which allowed us to query the function of the prototypical vacuole membrane protein EXP1.\n\nKnockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and genome editing in P. falciparum.

microbiology↗

bioRxiv2019

Arabidopsis SWR1-associated protein methyl-CpG-binding domain 9 is required for histone H2A.Z deposition.

Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the Arabidopsis SWR1-C has not been thoroughly characterized. Here identify interacting partners of a conserved Arabidopsis SWR1 subunit, ACTIN-RELATED PROTEIN 6 (ARP6). We isolated nine predicted components, and identified additional interactors implicated in histone acetylation and chromatin biology. One of the novel interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacted with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 was required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 was preferentially bound to nucleosome-depleted regions at the 5 ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes.

molecular biology↗