Biology subjects

Jaramillo, M.

Publications and source records attributed to Jaramillo, M..

4 recordsLinked to original sources

bioRxiv2022

Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates

Colonization of host phagocytic cells by Leishmania metacyclic promastigotes involves several parasite effectors, including the zinc-dependent metalloprotease GP63. The major mode of action of this virulence factor entails the cleavage/degradation of host cell proteins. Given the potent proteolytic activity of GP63, identification of its substrates requires the adequate preparation of cell lysates to prevent artefactual degradation during cell processing. In the present study, we re-examined the cleavage/degradation of reported GP63 substrates when GP63 activity was efficiently neutralized during the preparation of cell lysates. To this end, we infected bone marrow-derived macrophages with either wild type, {Delta}gp63, and {Delta}gp63+GP63 L. major metacyclic promastigotes for various time points. We prepared cell lysates in the absence or presence of the zinc-metalloprotease inhibitor 1,10-phenanthroline and examined the levels and integrity of ten previously reported host cell GP63 substrates. Inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the cleavage/degradation of several previously described GP63 targets, including PTP-PEST, mTOR, p65RelA, c-Jun, VAMP3, and NLRP3. Conversely, we confirmed that SHP-1, Synaptotagmin XI, VAMP8, and Syntaxin-5 are bona fide GP63 substrates. These results point to the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates. In addition, our results indicate that the role of GP63 in Leishmania pathogenesis must be re-evaluated. AUTHORS SUMMARYIn the protozoan parasite Leishmania, the abundant zinc-dependent metalloprotease GP63 is expressed at high levels at the surface of the promastigotes forms of the parasite. Upon phagocytosis by host macrophages, this metalloprotease is released from the parasites surface and spreads across the cytosol of infected cells. There, GP63 cleaves a number of host cell proteins involved in the control of host microbicidal function and in the regulation of immune responses, thereby contributing the ability of Leishmania to impair host defence mechanisms against infection. Given the abundance and powerful proteolytic activity of GP63, it is crucial to prevent artefactual proteolysis during processing of infected cells to identify genuine GP63 substrates. In this study, we found that inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the degradation of several of previously identified GP63 substrates. These results uncover the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates.

microbiology↗

bioRxiv2021

Cap-dependent translation initiation monitored in living cells

Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.

cell biology↗

bioRxiv2019

Translational profiling of macrophages infected with Leishmania donovani identifies mTOR- and eIF4A-sensitive immune-related transcripts

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. While previous studies showed that L. donovani reprograms transcription to subvert host cell functions, it remains unclear whether the parasite also alters host mRNA translation to establish a successful infection. To assess this, we compared transcriptome-wide translation in primary mouse macrophages infected with L. donovani promastigotes or amastigotes using polysome-profiling. This identified ample selective changes in translation (3,127 transcripts) which were predicted to target central cellular functions by inducing synthesis of proteins related to chromatin remodeling and RNA metabolism while inhibiting those related to intracellular trafficking and antigen presentation. Parallel quantification of protein and mRNA levels for a set of transcripts whose translation was activated upon L. donovani infection (Papbpc1, Eif2ak2, and Tgfb) confirmed, as indicated by polysome-profiling, increased protein levels despite largely unaltered mRNA levels. Mechanistic in silico analyses suggested activated translation depending on the kinase mTOR (e.g. Pabpc1) and the RNA helicase eIF4A (e.g. Tgfb) during infection. Accordingly, treatment with mTOR inhibitors torin-1 or rapamycin reversed L. donovani-induced PABPC1 without affecting corresponding transcript levels. Similarly, the production of TGF-{beta} decreased in presence of the eIF4A inhibitor silvestrol despite unaltered Tgfb mRNA levels. Consistent with parasite modulation of host eIF4A-sensitive translation to promote infection, silvestrol suppressed L. donovani replication within macrophages. In contrast, parasite survival was favored under mTOR inhibition. In summary, infection-associated changes in translation of mTOR- and eIF4A-sensitive mRNAs contribute to modulate mRNA metabolism and immune responses in L. donovani-infected macrophages. Although the net outcome of such translation programs favours parasite propagation, individual translation programs appear to have opposing roles during L. donovani infection, thereby suggesting their selective targeting as key for therapeutic effects. Author SummaryFine-tuning the efficiency of mRNA translation into proteins allows cells to tailor their responses to stress without the need for synthesizing new mRNA molecules. It is well established that the protozoan parasite Leishmania donovani alters transcription of specific genes to subvert host cell functions. However, discrepancies between transcriptomic and proteomic data suggest that post-transcriptional regulatory mechanisms also contribute to modulate host gene expression programs during L. donovani infection. Herein, we report that one third of protein-coding mRNAs expressed in macrophages are differentially translated upon infection with L. donovani. Our computational analyses reveal that subsets of mRNAs encoding functionally related proteins share the same directionality of translational regulation, which is likely to impact metabolic and microbicidal activity of infected cells. We also show that upregulated translation of transcripts that encode central regulators of mRNA metabolism and inflammation is sensitive to the activation of mTOR or eIF4A during infection. Finally, we observe that inhibition of eIF4A activity reduces parasite survival within macrophages while selective blockade of mTOR has the opposite effect. Thus, our study points to a dual role for translational control of host gene expression during L. donovani infection and suggests that novel regulatory nodes could be targeted for therapeutic intervention.

immunology↗

bioRxiv2019

Induction of an alternative 5’ leader enhances translation of Inpp5e and resistance to oncolytic virus infection

Residual cell-intrinsic innate immunity in cancer cells hampers infection with oncolytic viruses. mRNA translation is an important component of innate immunity, yet the targeted cellular mRNAs remain ill-defined. We characterized the translatome of resistant murine \"4T1\" breast cancer cells infected with three of the most clinically advanced oncolytic viruses: Herpes Simplex virus 1, Reovirus and Vaccinia virus. Common among all three infections were translationally de-repressed mRNAs involved in ciliary homeostasis including Inpp5e, encoding an inositol 5-phosphatase that modifies lipid second messenger signalling. Translationally repressed in the uninfected condition, viral infection induced expression of an Inpp5e mRNA variant that lacks repressive upstream open reading frames (uORFs) within its 5 leader and is consequently efficiently translated. Furthermore, we show that INPP5E contributes to antiviral immunity by altering virus attachment. These findings uncover a role for translational control through alternative 5 leader expression and assign ciliary proteins such as INPP5E to the cellular antiviral response.

molecular biology↗