Arnold C. Satterthwait

Humanin peptide suppresses apoptosis by interfering with Bax activation

Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

Mechanism of Bcl-2 and Bcl-X(L) inhibition of NLRP1 inflammasome: loop domain-dependent suppression of ATP binding and oligomerization.

NLRP1 (NLR family, pyrin domain-containing 1) is a contributor to innate immunity involved in intracellular sensing of pathogens, as well as danger signals related to cell injury. NLRP1 is one of the core components of caspase-1-activating platforms termed “inflammasomes,” which are involved in proteolytic processing of interleukin-1β (IL-1β) and in cell death. We previously discovered that anti-apoptotic proteins Bcl-2 and Bcl-XL bind to and inhibit NLRP1 in cells. Using an in vitro reconstituted system employing purified recombinant proteins, we studied the mechanism by which Bcl-2 and Bcl-XL inhibit NLRP1. Bcl-2 and Bcl-XL inhibited caspase-1 activation induced by NLRP1 in a concentration-dependent manner, with Ki ≈ 10 nM. Bcl-2 and Bcl-XL were also determined to inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and Bcl-XL was demonstrated to interfere with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-XL, which are located between the first and second α-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-XL inhibit NLRP1.

Alpha-helix nucleation between peptides using a covalent hydrogen bond mimic

Elena Z. Dovalsantos, Edelmira Cabezas and Arnold C. Satterthwait Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA Constraining peptides to the conformations they occupy in native proteins provides a means for converting biologically inactive peptides to bioactive peptides. We have shown that a peptide can be induced to form a full length α-helix in water by attaching its carboxyl terminus to an nucleation site (NucSite) [1]. A NucSite constitutes one turn of an stabilized by a covalent hydrogen bond mimic. In the initial NucSite, a hydrazone link was used to replace a hydrogen bond formed between a main-chain amide proton on one amino acid and an amide carbonyl oxygen of a second amino acid. In this work, we extend the utility of the NucSite by adding an α-amino group to the linker that allows its insertion between two peptides. In this case, the hydrazone link replaces a hydrogen bond that forms between the side chain of asparagine and the main chain amide proton of an (i, i +3) amino acid (Fig. 1). This design satisfies complex steric and stereochemical demands by building on a natural mechanism for a-helix nucleation used in proteins [2].