Research
The Cate Lab explores how genes are put into action by translation and also works on strategies for making new sequence-defined polymers using engineered ribosomes with C-GEM.
Eukaryotic Initiation Factor 3 (eIF3)
Protein synthesis –or translation– happens in three overall steps: initiation, elongation, and termination. In all of life, translation initiation is heavily regulated. That's probably because it's better not to start making a protein until it's needed, rather than stopping in the middle of making it. In humans, translation initiation involves many general translation factors, proteins and protein complexes called eukaryotic translation initiation factors or eIFs. We have focused on eIF3, because its large size remains a mystery. Human eIF3 is targeted by viruses like the hepatitis C virus that highjack translation for their own ends. We think that these viruses are tapping in to specific ways that eIF3 is used in normal human biology. With this idea in mind, our lab discovered that eIF3 is more than a general translation initiation factor. It can activate or repress translation of specific mRNAs that control how cells grow and divide. Notably, a number of these mRNAs encode key proteins involved in cancer. We're now working to understand how eIF3 binds these mRNAs in order to regulate their translation. We know that RNA structure is likely involved, rather than just a linear sequence pattern. For example, our lab has found that eIF3 binds to a stem loop in the 5’UTR of cJUN and BTG1. Interestingly, subunit d of eIF3 (eIF3d) also binds to the cap of cJUN, which suggests an eIF3d-directed cap-dependent mRNA translation which is alternative to the canonical eIF4E-dependent cap binding. Moreover, our lab has shown that eIF3 adds an additional layer of post-transcriptional regulation to highly regulated mRNAs such as FTL. Also, our lab has found that eIF3’s interaction with the PTBP1 mRNA is isoform and cell cycle dependent. These findings show the complexity of eIF3’s role in regulating translation of its target mRNAs, as well as the fact that these interactions are both mRNA and context specific. Because of this, we are also interested to find out if eIF3 regulates other mRNAs in different kinds of cells. So far, our lab has found that eIF3 target mRNAs differ between cell types. For example, in activated T-cells eIF3 binds to and regulates translation of mRNAs involved in T-cell activation and other mRNAs previously not found as eIF3 targets in human embryonic kidney cells. We are currently working on dissecting the mechanisms of these interactions and their relevance for human biology. We are also interested in deciphering the role of eIF3’s interactions with mRNAs involved in other processes occurring in these different cell types, such as in DNA damage, gene expression regulation, amongst others. As a whole, our goal is to understand the role of the outstandingly large, complex, and pivotal eIF3 more thoroughly.
The core of eIF3
(Sun et al.)
Ribosome Nascent Chain
Many human diseases are mediated by the activity of proteins. The convenient way to treat such diseases is by blocking the active site of harmful protein with selective small-molecule inhibitors. Although such approach has been widely and successfully used, some proteins may not have defined binding sites and remain “undruggable”. The alternative strategy to low down the harmful effect of such proteins is to affect their expression. The human ribosome plays a critical role in protein biosynthesis. Therefore, it is an attractive target for potential small-molecule inhibitors. Such molecules may selectively attenuate the expression of the protein of interest but do not possess the harmful cellular side-effects, mediated by general translation inhibition.
In our lab, we showed the PF-846 and chemically related compounds act as the mRNA-specific inhibitors of the previously undruggable proprotein convertase subtilisin/kexin type 9 (PCSK9), which plays a key role in cardiovascular diseases development by regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Using the ribosome profiling and biochemical approaches, it was shown that PF-846 highly selectively induces nascent-peptide mediated human ribosome stalling at codon 34 of PCSK9 mRNA as well as on a few other human transcripts. Cryo-electron microscopy experiments revealed the PF846 binding side, formed by an eukaryotic-specific pocket in the ribosome exit tunnel. Our studies show that interactions between PCSK9 nascent peptide and PF-846 molecule allosterically affect the docking of the reactive substrates in the ribosome active site. The selection of ribosome stalled complexes from in vitro translation system programmed by randomized mRNA libraries, combined with other biochemical experiments, further expand the spectrum of PF-846 mediated mRNA-specific effects. PF846 can stall not only ribosome at the elongation stage of translation but also arrest translation termination by inhibition of peptidyl-tRNA hydrolysis or even enhance translation, depending on nascent chain sequence context.
Even though the general principles of the transcript-specific mode of action of PF-846 and related compounds were not yet fully understood, their studies already have been laying the foundation for new therapeutic strategies.
Engineered Ribosomes
We are working with the Center for Genetically Encoded Materials to engineer bacterial ribosomes to make new kinds of polymers, rather than proteins. We study how ribosome structure and function can be altered to enable new polymerization chemistry in the ribosome. We are interested in designing screens for new activity, characterizing mutant ribosomes in vitro and with cryo-EM, and in gaining a better understanding of how the ribosome is (and is not) amenable to engineering. We are approaching this problem by understanding the scope of chemistry that can occur in the ribosome active site, the peptidyltransferase center, and identifying hurdles that exist to polymerize non-⍺-amino acids. Additionally we are developing methods to improve the stability and orthogonality of E. coli ribosomes for use in ribosome engineering.
Stem Cells
Stem cells exhibit a substantial increase in protein synthesis when they start differentiating towards progenitor cells. Inspired by previous studies from the Cate lab that have uncovered non-canonical roles of eIF3 in regulating protein translation in HEK293T and activated T cells, this project aims to decipher the specific molecular roles of eIF3 in neural stem cell differentiation. Below is a photo of differentiating Neural Stem Cells.
Car-T Cells
Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3’-untranslated regions (3’-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3’-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.
Model for robust T cell activation and improved CAR T cell function mediated by eIF3 interactions with the TCRA and TCRB mRNAs. (De Silva et al.)