4 research outputs found
INF2 is an endoplasmic reticulum-associated formin protein
In addition to its ability to accelerate filament assembly, which is common
to formins, INF2 is a formin protein with the unique biochemical ability to
accelerate actin filament depolymerization. The depolymerization activity of
INF2 requires its actin monomer-binding WASP homology 2 (WH2) motif. In this
study, we show that INF2 is peripherally bound to the cytoplasmic face of the
endoplasmic reticulum (ER) in Swiss 3T3 cells. Both endogenous INF2 and
GFP-fusion constructs display ER localization. INF2 is post-translationally
modified by a C-terminal farnesyl group, and this modification is required for
ER interaction. However, farnesylation is not sufficient for ER association,
and membrane extraction experiments suggest that ionic interactions are also
important. The WH2 motif also serves as a diaphanous autoregulatory domain
(DAD), which binds to the N-terminal diaphanous inhibitory domain (DID), with
an apparent dissociation constant of 1.1 μM. Surprisingly, the DID-DAD
interaction does not inhibit the actin nucleation activity of INF2; however,
it does inhibit the depolymerization activity. Point mutations to the DAD/WH2
inhibit both the DID-DAD interaction and depolymerization activity. Expression
of GFP-INF2 containing these DAD/WH2 mutations causes the ER to collapse
around the nucleus, with accumulation of actin filaments around the collapsed
ER. This study is the first to show the association of an actin-assembly
factor with the ER
Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice
Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance