Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase.

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsible for binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for the LHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle.

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes.

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates.

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.

A dynamic cpSRP43-Albino3 interaction mediates translocase regulation of chloroplast signal recognition particle (cpSRP)-targeting components.

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.

First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers.

This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17-19 d in the 3 highest dose groups and volume of distribution of 5-6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use.

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.

Radiation-induced galectin-1 by endothelial cells: a promising molecular target for preferential drug delivery to the tumor vasculature.

The present study reports on a new strategy for selective, radiation therapy-amplified drug delivery using an antiangiogenic 33-a.a., tumor vasculature-targeting ligand, anginex, to improve the therapeutic ratio for strategies developed against solid tumors. Our findings indicate that galectin-1 is (a) one of the major receptors for anginex (b) overexpressed by tumor neovasculature and (c) further specifically upregulated in endothelial cells in response to radiation exposure as low as 0.5 Gy. An investigation of [18]-F-labeled anginex biodistribution in SCK tumors indicates that anginex is an effective targeting molecule for image and radiation-guided therapy of solid tumors. An anginex-conjugated liposome capable of being loaded with drug was shown to selectively target endothelial cells post-radiation. The presence of endothelial cells in a three-dimensional co-culture system with tumor cells developed to study tumor/endothelial cell interactions in vitro led to higher levels of galectin-1 and showed a further increase in expression upon radiation exposure when compared to tumor cell spheroids alone. Similar increase in galectin-1 was observed in tumor tissue originating from the tumor-endothelial cell spheroids in vivo and radiation exposure further induced galectin-1 in these tumors. The overall results suggest feasibility of using a clinical or subclinical radiation dose to increase expression of the galectin-1 receptor on the tumor microvasculature to promote delivery of therapeutics via the anginex peptide. This approach may reduce systemic toxicity, overcome drug resistance, and improve the therapeutic efficacy of conventional chemo/radiation strategies.

The membrane-binding motif of the chloroplast signal recognition particle receptor (cpFtsY) regulates GTPase activity.

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.

Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse.

Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential. In this minireview we discuss some of the antibody forms that may be effective clinical treatments for drug abuse, as well as several current and emerging production systems that could bridge the gap from discovery to patient use.

Deletion of the chloroplast-localized Thylakoid formation1 gene product in Arabidopsis leads to deficient thylakoid formation and variegated leaves.

Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. The Arabidopsis (Arabidopsis thaliana) Thylakoid formation1 (Thf1) gene product is shown here to control an important step required for the normal organization of these vesicles into mature thylakoid stacks and ultimately for leaf development. The Arabidopsis Thf1 gene encodes an imported chloroplast protein, as shown by in vitro import and localization of a Thf1-green fluorescent protein fusion product in transgenic plants. This gene is conserved in oxygenic photoautotrophs ranging from cyanobacteria to flowering land plants. Transcript levels for Thf1 are induced in the light and decrease under dark conditions, paralleling profiles of light-regulated nuclear genes involved in chloroplast function. Disruption of the Thf1 gene via T-DNA insertion results in plants that are severely stunted with variegated leaf patterns. Nongreen sectors of variegated leaves lacking Thf1 expression contain plastids that accumulate membrane vesicles on the interior and lack organized thylakoid structures. Green sectors of Thf1-disrupted leaves contain some chloroplasts that form organized thylakoid membranes, indicating that an inefficient compensatory mechanism supports thylakoid formation in the absence of Thf1. Genetic complementation of a Thf1 knockout line confirms the role of this gene in chloroplast and leaf development. Transgenic plants expressing the Thf1 gene in antisense orientation are stunted with altered thylakoid organization, especially in young seedlings. The data indicate that the Thf1 gene product plays a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis.

Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43.

The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.