Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.Peer reviewe

Functional Analysis of Replication Focus Targeting Sequence Mutations in DNA Methyltransferase 1

The addition of a methyl group to cytosine at CpG sites in DNA is a major epigenetic mechanism in humans. Faithful replication of epigenetic markers through successive rounds of DNA replication is crucial for preservation of cell specific gene expression. Failure to maintain methylation patterns, the primary role of human DNA methyltransferase 1 (DNMT1), has been linked to the development of a spectrum of neurodegenerative disorders. Specifically, mutations in the regulatory replication focus targeting sequence (RFTS) domain of DNMT1 have been linked to the neurodegenerative disorder hereditary sensory and autonomic neuropathy type 1E (HSAN1E). HSAN1E is characterized by sensory neuropathy, hearing loss, and cognitive decline, on average presenting at 37.7 years of age with survival to 53.6 years of age. The objective of this thesis is to investigate how three RFTS point mutants (C353F, P491L, and I531N) identified in HSAN1E kinships contribute to the associated phenotype of global hypomethylation and site-specific hypermethylation. The RFTS domain is known to act in an autoinhibitory manner, binding the catalytic domain and preventing DNA binding; we hypothesized that proteins containing RFTS mutations would have decreased overall stability and partial relief of autoinhibition leading to increased activity. Two of the mutant proteins (C353F and I531N) were purified to homogeneity; the third (P491L) was not soluble. The purified enzymes exhibited increased heparin affinity, reduced thermal stability, and increased DNA binding activity as compared to the wildtype enzyme. The mutant C353F also exhibited increased temperature-dependent aggregate formation. Combined, these results support the claim that these disease-causing mutations in the RFTS domain result in proteins that are less stable but more active than wildtype DNMT1, contributing to a lab-wide effort to better understand the molecular mechanisms of disease formation

Ghrelin Receptor (GHS-R1A) Antagonism Alters Preference for Ethanol and Sucrose in a Concentration-Dependent Manner in Prairie Voles

Ghrelin receptor (GHS-R1A) activity has been implicated in reward for preferred foods and drugs; however, a recent study in our laboratory indicated that GHS-R1A antagonism reduces early (after only four exposures) preference for 20% ethanol, but not 10% sucrose in prairie voles, a genetically diverse high alcohol-consuming species. The purpose of the present study was to determine if these effects of GHS-R1A antagonism depend on the concentration of the rewarding solution being consumed. We first characterized preference for varying concentrations of ethanol and sucrose. Two bottle tests of each ethanol concentration versus water indicated that 10% and 20% ethanol are less preferred than 3% ethanol, and a follow-up direct comparison of 10% vs. 20% showed that 10% was preferred over 20%. Direct two-bottle comparisons of 2% vs. 5%, 2% vs. 10%, and 5% vs. 10% sucrose showed that 10% sucrose was most preferred, and 2% sucrose was least preferred. The effects of JMV 2959, a GHS-R1A antagonist, on preference for each concentration of ethanol and sucrose were then tested. In a between groups design prairie voles were given four two-hour drinking sessions in which animals had access to ethanol (3, 10, or 20%) versus water, or sucrose (2, 5, or 10%) versus water every other day. Saline habituation injections were given 30min before the third drinking session. JMV 2959 (i.p.; 9mg/kg), a GHS-R1A antagonist, or saline was administered 30min before the fourth drinking session. JMV 2959 reduced preference for 20% ethanol and 2% sucrose, but had no significant effect on preference for the other ethanol and sucrose concentrations. These data identify constraints on the role of GHS-R1A in early preference for ethanol and sucrose, and the concentration-dependent effects suggest strong preference for a reward may limit the importance of GHS-R1A activity.Copyright 2015 Elsevier Inc. All rights reserved