Madan M. Paidhungat

Yeast Mn2+ transporter, Smf1p, is regulated by ubiquitin-dependent vacuolar protein sorting.

Conditional cdc1(Ts) mutants of S. cerevisiae arrest with a phenotype similar to that exhibited by Mn2+-depleted cells. Sequence similarity between Cdc1p and a class of Mn2+-dependent phosphoesterases, as well as the observation that conditional cdc1(Ts) growth can be ameliorated by Mn2+ supplement, suggests that Cdc1p activity is sensitive to intracellular Mn2+ levels. This article identifies several previously uncharacterized cdc1(Ts) suppressors as class E vps (vacuolar protein sorting) mutants and shows that these, as well as other vps mutants, accumulate high levels of intracellular Mn2+. Yeast VPS genes play a role in delivery of membrane transporters to the vacuole for degradation, and we show that the vps mutants accumulate elevated levels of the high-affinity Mn2+ transporter Smf1p. cdc1(Ts) conditional growth is also alleviated by mutations, including doa4 and ubc4, that compromise protein ubiquitination, and these ubiquitination defects are associated with Smf1p accumulation. Epistasis studies show that these suppressors require functional Smf1p to alleviate the cdc1(Ts) growth defect, whereas Smf1p is dispensable for cdc1(Ts) suppression by a mutation (cos16/per1) that does not influence intracellular Mn2+ levels. Because Smf1p is ubiquitinated in vivo, we propose that Smf1p is targeted to the vacuole for degradation by ubiquitination-dependent protein sorting.

Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.

ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution

The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials.