Ravishankar, R. | Search bioRxiv

bioRxiv2023

A Humanized Yeast System for Evaluating the Protein Prenylation of a Wide Range of Human and Viral CaaX Sequences

The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur in 1-2% of proteins within yeast and human proteomes. Recently, non-canonical CaaX sequences in addition to shorter and longer length CaX and CaaaX sequences have been identified that can be prenylated. Much of the characterization of prenyltransferases has relied on the yeast system because of its genetic tractability and availability of reporter proteins, such as the a-factor mating pheromone, Ras GTPase, and Ydj1 Hsp40 chaperone. To compare the properties of yeast and human prenyltransferases, including the recently expanded target specificity of yeast farnesyltransferase, we have developed yeast strains that express human farnesyltransferase or geranylgeranyltransferase-I in lieu of their yeast counterparts. The humanized yeast strains display robust prenyltransferase activity that functionally replaces yeast prenyltransferase activity in a wide array of tests, including the prenylation of a wide variety of canonical and non-canonical human CaaX sequences, virus encoded CaaX sequences, non-canonical length sequences, and heterologously expressed human proteins HRas and DNAJA2. These results reveal highly overlapping substrate specificity for yeast and human farnesyltransferase, and mostly overlapping substrate specificity for GGTase-I. This yeast system is a valuable tool for further defining the prenylome of humans and other organisms, identifying proteins for which prenylation status has not yet been determined. Summary StatementWe report yeast engineered to express human prenylation enzymes with which prenylation can be investigated for established and novel CaaX sequences associated with proteins involved in human disease.

biochemistry↗

bioRxiv2022

Therapeutic Interruption of T Cell Development Generates High-Affinity T Cells That Escape Exhaustion and Improve Cancer Immunotherapy

Availability of effective anti-tumor T cells is limited by cancer immunoediting, which depletes neoantigens, and central tolerance, which eliminates developing T cells with high-affinity T cell receptors (TCRs) against tumor self-antigens. Remaining tumor-reactive T cells are often exhausted after immune checkpoint blockade (ICB). Whether endogenous T cells with high- affinity TCRs against tumor self-antigens can be generated to circumvent exhaustion and reject neoantigen-poor tumors is unclear. We show that transiently interrupting central tolerance through RANKL blockade unleashes T cells possessing TCRs with self-reactive features that enable ICB to reject poorly immunogenic tumors. Upon recognition of tumor self-antigens, these T cells exhibit enhanced TCR signaling, enrichment in NFAT/AP-1 genes, and lymph node priming. Consequently, memory-precursor T cells against tumor self-antigens are generated, avoid exhaustion, and become effector-memory cells with transcriptional features associated with clinical ICB response. Thus, interrupting central tolerance provides T cells with tumor- directed autoreactivity that avoid exhaustion and improve immunotherapy.

immunology↗