Biology subjects

Blakely, W. J.

Publications and source records attributed to Blakely, W. J..

4 recordsLinked to original sources

bioRxiv2024

The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii

Intracellular replication is crucial for the success of apicomplexan parasites, including Toxoplasma gondii. Therefore, essential players in parasite replication present potential targets for drug development. In this study, we have characterized TgGSK, a glycogen synthase kinase homolog that plays an important role in Toxoplasma endodyogeny. We have shown that TgGSK has a dynamic localization that is concurrent with the cell cycle. In non-dividing parasites, this kinase is highly concentrated in the nucleus. However, during division, TgGSK displays a cytosolic localization, with concentration foci at the centrosomes, a key organelle involved in parasite division, and the basal end. Conditional knockdown of TgGSK determined that it is essential for the completion of the lytic cycle and proper parasite division. Parasites lacking endogenous protein levels of TgGSK exhibited defects in division synchronicity and the segregation of the nucleus and apicoplast into forming daughter cells. These phenotypes are associated with defects in centrosome duplication and fission. Global phosphoproteomic analysis determined TgGSK-dependent phosphorylation of RNA-processing, basal end, and centrosome proteins. Consistent with the putative regulation of RNA-processing proteins, global transcriptomic analysis suggests that TgGSK is needed for proper splicing. Finally, we show that TgGSK interacts with GCN5b, a well-characterized acetyltransferase with roles in transcriptional control. Conversely, GCN5b chemical inhibition results in specific degradation of TgGSK. Thus, these studies reveal the involvement of TgGSK in various crucial processes, including endodyogeny and splicing, and identify acetylation as a possible mechanism by which this essential kinase is regulated. AUTHOR SUMMARYToxoplasma gondii infects nearly a third of the worlds human population. While infection is largely asymptomatic in healthy adults, in immunocompromised or immunosuppressed individuals it can lead to brain lesions and even death. Similarly, toxoplasmosis can result in stillbirth, birth defects, and blindness of a developing fetus in the case of a congenital infection. With minimal treatments for Toxoplasmosis available, it is crucial to study parasite-specific processes that could be potential drug targets for the treatment of Toxoplasmosis. In this study, we investigated the protein TgGSK that is essential for parasite survival and proper division. We showed that TgGSK may perform its essential functions through interaction with the centrosome, an organelle that plays a major role in cell division in many organisms. We also show in this study a role for TgGSK in proper processing of messenger RNAs. Taken together, we have performed an in-depth study of the functional role of the essential protein TgGSK in Toxoplasma gondii. Importantly, TgGSK was shown to have more similarity to plant proteins than mammalian proteins which may allow for the possibility of targeting of this protein for therapeutic treatment of toxoplasmosis.

microbiology↗

bioRxiv2024

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active site were required for PTPN14 to promote differentiation. Taken together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation. SIGNIFICANCEThe Hippo kinase cascade inhibits YAP1, an oncoprotein and driver of cell stemness and self-renewal. There is mounting evidence that the Hippo pathway is targeted by tumor viruses including human papillomavirus. The high-risk HPV E7 oncoprotein promotes YAP1 nuclear localization and the carcinogenic activity of high-risk HPV E7 requires YAP1 activity. Blocking HPV E7-dependent YAP1 activation could inhibit HPV-mediated carcinogenesis, but the mechanism by which HPV E7 activates YAP1 has not been elucidated. Here we report that by degrading the tumor suppressor PTPN14, HPV18 E7 inhibits LATS1 kinase, reducing inhibitory phosphorylation on YAP1. These data support that an HPV oncoprotein can inhibit Hippo signaling to activate YAP1 and strengthen the link between PTPN14 and Hippo signaling in human epithelial cells.

microbiology↗

bioRxiv2022

The tyrosine phosphatase PRL regulates attachment of Toxoplasma gondii to host cells and is essential for virulence

The pathogenesis of Toxoplasma gondii is mainly due to tissue damage caused by the repeating lytic cycles of the parasite. Many proteins localized to the pellicle of the parasite, particularly kinases, have been identified as critical regulators of the Toxoplasma lytic cycle. However, little is known about the associated protein phosphatases. Phosphatase of regenerating liver (PRL), a highly conserved tyrosine phosphatase, is an oncogene that plays pivotal roles in mammalian cells and typically associates with membranes via a conserved prenylation site. PRL in Toxoplasma has a predicted prenylation motif in the C-terminus like other homologs. We have determined that TgPRL localizes to the plasma membrane and that disruption of TgPRL results in a defect in the parasites ability to attach to host cells. This function is dependent both on TgPRLs membrane localization and phosphatase activity. Importantly, in vivo experiments have shown that while mice infected with parental strain parasites die within days of infection, those infected with parasites lacking TgPRL not only survive but also develop immunity that confers protection against subsequent infection with wild-type parasites. Immunoprecipitation experiments revealed that the PRL-CNNM (Cyclin M) complex, which regulates intracellular Mg2+ homeostasis in mammalian cells, is also present in Toxoplasma. Consistent with this interaction, parasites lacking TgPRL had higher intracellular Mg2+ levels than the parental or complemented strains, suggesting TgPRL is involved in regulating intracellular Mg2+ homeostasis. Thus, TgPRL is a vital regulator of the Toxoplasma lytic cycle and virulence, showing its potential as a target of therapeutic intervention. IMPORTANCEInfection with Toxoplasma gondii can lead to severe and even life-threatening diseases in people with compromised or suppressed immune systems. Unfortunately, drugs to combat the parasite are limited, highly toxic, and ineffective against the chronic stage of the parasite. Consequently, there is a strong demand for the discovery of new treatments. A comprehensive understanding of how the parasite propagates in the host cells and which proteins contribute to the parasites virulence will facilitate the discovery of new drug targets. Our study meets this objective and adds new insights to understanding the lytic cycle regulation and virulence of Toxoplasma by determining that the protein phosphatase TgPRL plays a vital role in the parasites ability to attach to host cells and that it is essential for parasite virulence.

microbiology↗

bioRxiv2019

The secreted acid phosphatase domain-containing GRA44 from Toxoplasma gondii is required for C-myc induction in infected cells

During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parsitophorous vacuole to safely reside within, while partitioned from host cell defense mechanisms. From within this safe niche parasites sabotage multiple host cell systems including gene expression, apoptosis and intracellular immune recognition by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. Translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins included MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell cMyc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCEApproximately one third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. To replicate Toxoplasma requires to enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

microbiology↗