Brief Summaries of Manuscripts Presented/Reviewed at Lab Meeting
2023 06 01 - Adam presented a paper titled "Design of Synthetic Promoters for Cyanobacteria with Generative Deep-Learning Model" from Nucleic Acids Research
Seo et al. have developed a deep-learning-based framework by which they were able to design and predict the effectiveness of synthetic promoters for a species of cyanobacteria with limited genetic tools available. The authors used a generational deep-learning model known as a 'variational autoencoder' (VAE) that they trained with ~ 4,000 putative promoters from RNA-sequencing data. The effectiveness of the sequences that their VAE generated were then evaluated using a second model, a convolutional neural network, which was also trained on RNA-sequencing data. The authors confirmed that their models were capturing meaningful features from their training data and went on to validate a handful of synthetic promoters in cell-free transcription/translation assays and in vivo YFP expression assays. In short, the step-wise method proposed by Seo et al. could provide a robust foundation for the rapid in silico generation and in vitro/in vivo evaluation of synthetic promoter sequences -- especially in non-model organisms (like Synechocystis).
2023 06 15 - Matt presented a paper titled "A Spatiotemporal Notch Interaction Map from Membrane to Nucleus" from Biorxiv
Martin et al. utilized Notch2 (a foundational protein in the Gordon Lab) fused with APEX a protein that biotinylates proximal proteins. The researchers utilized this construct following treatment and washout of Notch2 inhibitors at different time points allowing for the identification of the proteins Notch2 interacts discrete points as the receptor is engaged, proteolyzed, and the intracellular domain (ICD) is trafficked to the nucleus. This work demonstrated that the ICD is released following endocytosis and then is rapidly shuttled into the nucleus. This shuttling happened in the presence of cytoskeleton proteins, yet these proteins may not be essential following knockdown of the three most common proteins the ICD still enters the nucleus. Once in the nucleus first interacts with chromatin remodeling proteins and then RNA binding proteins. This work established the spatiotemporal map of Notch associated proteins and offers insights into potential regulatory mechanisms the Gordon Lab is excited to pursue.
2023 07 03 - Mike presented a paper titled “Bacterial SEAL domains undergo autoproteolysis and function in regulated intramembrane proteolysis” from Biorxiv
Brogan et al. identify a sea urchin sperm protein, enterokinase, agrin (SEA)-like domain (SEAL domain) in a bacterial protein, RsgI. This domain is common in proteins that are sensitive to mechanical force, including many that the Gordon lab studies. They use both AlphaFold and x-ray crystallography to show that this protein exhibits a similar fold and similar characteristics to identified SEA proteins, including torsional strain on one loop which induces autoproteolysis. RsgI requires mechanical force to be proteolyzed which induces downstream signaling cascades. These proteins evolved separately from mammalian SEA domains even though many features are shared between them which begs the question about why this domain fold is found in so many mechanoreceptors.
2023 08 03 - Adam presented a paper titled "Accessing Unexplored Regions of Sequence Space in Directed Enzyme Evolution Via Insertion/Deletion Mutagenesis" from Nature Communications
Emond et al. have developed a novel pair of transposase-based mutagenesis methods that enable single, double, or triple deletions or insertions of nucleotide triplets for protein engineering efforts. Their methods, TransDel and TransIn, either delete or insert random strings of three, six, or nine nucleotides, respectively, in order to generate mutagenic libraries to explore the often-overlooked sequence space of deletions and insertions. Historically, protein engineering efforts have been limited to simple substitutions through either error-prone PCR or saturation mutagenesis with degenerate codons. While substitutions do play a role in protein evolution, they act in concert with other factors like recombination, and, importantly, insertions/deletions in order to drive the development of novel function. The authors find that their novel methods are able to effectively access this unexplored sequence space, and that the vast majority of mutations induced from both TransDel and TransIn are strongly deleterious. Moreover, though they find that most of this new sequence space is functionally sparse they also note that beneficial mutations were much more common in their insertion and deletion libraries than in their substitution libraries, indicating that their is great potential value in this underexplored area of evolution.
2023 08 15 – Natalia presented a paper titled “High-throughput characterization of HLA-E-presented CD94/NKG2x ligands reveals peptides which modulate NK cell activation” from Nature Communications
Huisman et al. developed a high-throughput method by combining yeast-display and prediction algorithms to screen a large peptide library to identify peptides that bind HLA-E and CD94/NKG2A or CD94/NKG2C. Human leukocyte antigen E (HLA-E) is a class 1b Major Histocompatibility Complex (MHC) expressed by most cells that binds signal peptides which are recognized by the receptors CD94/NKG2x in NK cells. When these are downregulated by pathogens, NK cells initiate cytotoxic activity. These receptors are expressed differentially within patients and diseases, making their modulation of therapeutic interest. Since there is a limited number of known HLA-E peptide binders, Huisman et al. developed a framework to study and characterize a library of randomized peptides to identify binders and possibly use these for future peptide therapeutics. They designed and screened a library of peptides for yeast surface display that would bind both the HLA-E and the cognate receptors CD94/NKG2x in a high-affinity manner. Since the peptide library has a large theoretical sequence space, they trained a prediction algorithm with the sequencing data from their yeast display selections and identified human and cytomegalovirus (CMV) HLA-E presented peptides that are capable of binding. They then continued to characterize the binding of these peptides to HLA-E by assesing their capability of stabilizing HLA-E on cells and found strong stabilization of HLA-E by ten human peptides. They continued to study how these predicted peptides could modulate NK cell activation by interacting with the cognate receptors CD94/NKG2x in cell-based activation assays and found that six peptides had strong effects on NK modulation, whereas four of the peptides had either moderate or strong NK modulation. The results shown by this research shows a promising and innovative way of modulating NK cell activity which would be beneficial for future therapeutics.