Structural analysis of chloroplast tail‐anchored membrane protein recognition by ArsA1

In mammals and yeast, tail‐anchored (TA) membrane proteins destined for the post‐translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well‐known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane‐trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide‐free open state and bound to adenylyl‐imidodiphosphate. This approximately 80‐kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA‐binding groove comprise the interlocking hook‐like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.     

CD40L reverse signaling suppresses prevertebral sympathetic axon growth and tissue innervation

CD40‐activated CD40L reverse signaling is a major physiological regulator of the growth of neural processes in the developing nervous system. Previous work on superior cervical ganglion (SCG) neurons of the paravertebral sympathetic chain has shown that CD40L reverse signaling enhances NGF‐promoted axon growth and tissue innervation. Here we show that CD40L reverse signaling has the opposite function in prevertebral ganglion (PVG) sympathetic neurons. During a circumscribed perinatal window of development, PVG neurons cultured from Cd40^–/– mice had substantially larger, more exuberant axon arbors in the presence of NGF than PVG neurons cultured from wild‐type mice. Tissues that receive their sympathetic innervation from PVG neurons were markedly hyperinnervated in Cd40^–/– mice compared with wild‐type mice. The exuberant axonal growth phenotype of cultured CD40‐deficient perinatal PVG neurons was pared back to wild‐type levels by activating CD40L reverse signaling with a CD40‐Fc chimeric protein, but not by activating CD40 forward signaling with CD40L. The co‐expression of CD40 and CD40L in PVG neurons suggests that these proteins engage in an autocrine signaling loop in these neurons. Our work shows that CD40L reverse signaling is a physiological regulator of NGF‐promoted sympathetic axon growth and tissue innervation with opposite effects in paravertebral and prevertebral neurons.     

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.     

Coronin 3 involvement in F-actin-dependent processes at the cell cortex

The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin-associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3-mediated events.     

A loop of coagulation factor VIIa influencing macromolecular substrate specificity

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).     

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase alpha-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.     

Protective Effects of Vitamin E against Oxidative Damage Induced by Aβ1-40Cu（Ⅱ） Complexes

β-amyloid peptide (Aβ) is considered to be responsible for the formation of senile plaques,which is the hallmark of Alzheimer's disease (AD).Oxidative stress,manifested by protein oxidation andlipid peroxidation,among other alterations,is a characteristic of AD brain.A growing body of evidence hasbeen presented in support of Aβ_(1-40) forming an oligomeric complex that binds copper at a CuZn superoxidedismutase-like binding site. Aβ_(1-40)Cu(Ⅱ) complexes generate neurotoxic hydrogen peroxide (H_2O_2) from O_2via Cue reduction,though the precise reaction mechanism is unclear.The toxicity of Aβ_(1-40) or the Aβ_(1-40)Cu(Ⅱ)complexes to cultured primary cortical neurons was partially attenuated when ( )-α-tocopherol (vitamin E)as free radical antioxidant was added at a concentration of 100 μM.The data derived from lactate dehydro-genase (LDH) release and the formation of H_2O_2 confirmed the results from the MTT assay.These findingsindicate that copper binding to Aβ_(1-40) can give rise to greater production of H_2O_2, which leads to a break-down in the integrity of the plasma membrane and subsequent neuronal death.Groups treated with vitaminE exhibited much slighter damage,suggesting that vitamin E plays a key role in protecting neuronal cellsfrom dysfunction or death.     

1H, 13C, and 15N backbone resonance assignments of the carboxyl terminal domain of Connexin40

Connexin40 is a gap junction protein involved in cell communication in the heart and other tissues. The assignments of an important Connexin40 regulatory domain, the carboxyl terminus, will aid in identifying the types of inter- and intramolecular interactions that affect channel activity.