Biology subjects

Jami-Alahmadi, Y.

Publications and source records attributed to Jami-Alahmadi, Y..

25 records · Page 2Linked to original sources

bioRxiv2022

Substrate trapping approach identifies TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes

The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), and nucleoside synthesis (NME1). R54P abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in diverse biological processes.

cell biology↗

bioRxiv2021

The dynamics of HD-ZIP III - ZPR protein interactions play essential roles in embryogenesis, meristem function and organ development

Maintaining a stem cell population while developing lateral organs is a crucial aspect of plant development. Studies have shown that a family of micro proteins, the LITTLE ZIPPERS (ZPR), are involved in this process by repressing the activity of HD-ZIP III transcription factors. However, the unique role of each ZPR has not been thoroughly characterized. In this work, we use genetics, imaging, and biochemistry to create a detailed picture of ZPR family expression and their specific interactions with HD-ZIP IIIs. CRISPR/Cas9 was implemented to isolate single loss-of-function ZPR alleles as well as higher-order mutant combinations. A single mutation in ZPR1, ZPR3, and ZPR4 affects the development of the cotyledons during embryogenesis. Additionally, double mutant analyses indicates both unique roles for each ZPR protein as well as redundancy. Using ZPR tagged lines we show that while ZPR3 and ZPR4 have a similar pattern of accumulation as the HD-ZIP IIIs, ZPR1 and ZPR2 accumulation is more limited. Immuno-precipitations (IP) with tagged ZPR proteins are mainly enriched with the anticipated HD-ZIP III partners. Although ZPRs interact with all HD-ZIP IIIs, an apparent preference of heterodimer formation with REVOLUTA is observed. Our work highlights that the dynamics of ZPR protein accumulation together with the strength of ZPR-HD-ZIP III interactions provide an added layer of complexity to the regulation of HD-ZIP IIIs during plant development.

plant biology↗

bioRxiv2021

Divergent Acyl Carrier Protein Mediates Mitochondrial Fe-S Cluster Biosynthesis in Malaria Parasites

Plasmodium falciparum malaria parasites are early-diverging eukaryotes with many unusual metabolic adaptations. Understanding these adaptations will give insight into parasite evolution and unveil new parasite-specific drug targets. Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis. In yeast and humans, mACP also functions to biochemically couple FASII activity to electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast to most eukaryotes, the Plasmodium mitochondrion lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC followed by broader cellular defects. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of both Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other Apicomplexan pathogens, including Toxoplasma and Babesia. This discovery also highlights the ancient, fundamental role of ACP in mitochondrial Fe-S cluster biogenesis and unveils an evolutionary driving force to retain this interaction with ACP independent of its eponymous function in FASII. Significance StatementPlasmodium malaria parasites are single-celled eukaryotes that evolved unusual metabolic adaptations. Parasites require a mitochondrion for blood-stage viability, but essential functions beyond the electron transport chain are sparsely understood. Unlike yeast and human cells, the Plasmodium mitochondrion lacks fatty acid synthesis enzymes but retains a divergent acyl carrier protein (mACP) incapable of tethering acyl groups. Nevertheless, mACP is essential for parasite viability by binding and stabilizing the core mitochondrial Fe-S cluster biogenesis complex via a divergent molecular interface lacking an acyl-pantetheine group that contrasts with other eukaryotes. This discovery unveils an essential metabolic adaptation in Plasmodium and other human parasites that decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis and evolved at or near the emergence of Apicomplexan parasitism.

microbiology↗

bioRxiv2020

Activation of FAM111A Protease Induces Defects in Nuclear Function that Likely Underlie its Roles in Disease and Viral Restriction

Mutations in the nuclear trypsin-like serine protease FAM111A cause Kenny-Caffey syndrome (KCS2) with hypoparathyroidism and skeletal dysplasia, or perinatally lethal osteocraniostenosis (OCS). In addition, FAM111A was identified as a restriction factor for certain host range mutants of the SV40 polyomavirus and VACV orthopoxvirus. However, because FAM111A function is poorly characterized, its roles in restricting viral replication and the etiology of KCS2 and OCS remain undefined. We find that the FAM111A KCS2 and OCS patient mutants are hyperactive, inducing apoptosis-like phenotypes in a protease-dependent manner. Similarly, in response to the attempted replication of SV40 host range mutants in restrictive cells, FAM111A activity induces the loss of nuclear barrier function and structure. Interestingly, pan-caspase inhibitors do not block FAM111A-dependent phenotypes such as nuclear "leakiness", shrinkage and pore redistribution, implying it acts independently or upstream of caspases. In this regard, we identified nucleoporins and the associated GANP transcription factor as FAM111A interactors and candidate targets. Together our data provide key insight into how FAM111A activation can restrict viral replication, and how its deregulated activity could cause KCS2 and OCS.

cell biology↗

bioRxiv2020

Wnt-inducible Lrp6-APEX2 Interacting Proteins identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as {beta}-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of {beta}-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/{beta}-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling. SignificanceWnt/{beta}-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylated-enriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling.

cell biology↗

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

The plant mobile domain proteins MAIN and MAIL1 interact with the phosphatase PP7L to regulate gene expression and silence transposable elements in Arabidopsis thaliana.

Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs. AUTHOR SUMMARYThe plant mobile domain (PMD) is a protein domain of unknown function that is widely spread in the angiosperm plants. Although most PMDs are associated with repeated DNA sequences called transposable elements (TEs), plants have domesticated the PMD to produce genic versions that play important roles within the cell. In Arabidopsis thaliana, MAINTENANCE OF MERISTEMS (MAIN) and MAIN-LIKE 1 (MAIL1) are genic PMDs that are involved in genome stability, developmental processes, and silencing of TEs. The mechanisms involving MAIN and MAIL1 in these cellular processes remain elusive. Here, we show that MAIN, MAIL1 and the phosphoprotein phosphatase (PPP) named PP7-like (PP7L) interact to form a protein complex that is required for the proper expression of genes, and the silencing of TEs. Phylogenetic analyses revealed that PMD and PP7-type PPP domains are evolutionary connected, and several plant species express proteins carrying both PMD and PPP domains. We propose that interaction of PMD and PPP domains would create a functional protein module involved in mechanisms regulating gene expression and repressing TEs.

plant biology↗