Association of adaptor protein TRIP8b with clathrin

TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.     

Missing-in-metastasis protein promotes internalization of magnetic nanoparticles via association with clathrin light chain and Rab7

Background

Magnetic nanoparticles (MNPs) have been widely used in biomedical applications. Proper control of the duration of MNPs in circulation promises to improve further their applications, in particularly drug delivery. It is known that the uptake of tissue-associated MNPs is mainly carried out by macrophages. Yet, the molecular mechanism to control MNPs internalization in macrophages remains to be elusive. Missing-in-metastasis (MIM) is a scaffolding protein that is highly expressed in macrophages and regulates receptor-mediated endocytosis. We hypothesize that uptake of MNPs may also involve the function of MIM.

自组装在多肽药物中的应用

自组装是指分子、纳米级结构材料等基本单元自发地组装成一个稳定而又紧密结构的过程。多肽可在各种非共价驱动力下自组装形成纳米纤维、纳米层状结构、胶束等不同的形貌。因多肽具有氨基酸序列明确、易于合成、便于设计等优势,多肽自组装技术成为了近年来的一个研究热点。有研究表明,对某些多肽类药物进行自组装设计或者使用自组装肽材料作为药物递送的载体,可以解决药物自身存在的半衰期短、水溶性差、生理屏障穿透率低等问题。本文重点介绍了自组装多肽的形成机制、自组装形貌、影响因素、自组装设计方法及其在生物医学领域的主要应用,为多肽的高效利用提供参考。     

Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor,fibrinogen and fibronectin

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of -granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the -granules.     

Problems,successes and challenges for the application of dispersion-corrected density-functional theory combined with dispersion-based implicit solvent models to large-scale hydrophobic self-assembly and polymorphism

The recent advent of dispersion-corrected density-functional theory (DFT) methods allows for quantitative modelling of molecular self-assembly processes, and we consider what is required to develop applications to the formation of large self-assembled monolayers (SAMs) on hydrophobic surfaces from organic solution. Focus is on application of the D3 dispersion correction of Grimme combined with the solvent dispersion model of Floris, Tomasi and Pascual–Ahuir to simulate observed scanning-tunnelling microscopy (STM) images of various polymorphs of tetraalkylporphyrin SAMs on highly oriented pyrolytic graphite surfaces. The most significant problem is identified as the need to treat SAM structures that are incommensurate with those of the substrate, providing a challenge to the use of traditional periodic-imaging boundary techniques. Using nearby commensurate lattices introduces non-systematic errors into calculated lattice constants and free energies of SAM formation that are larger than experimental uncertainties and polymorph differences. Developing non-periodic methods for polymorph interface simulation also remains a challenge. Despite these problems, existing methods can be used to interpret STM images and SAM atomic structures, distinguishing between multiple feasible polymorph types. They also provide critical insight into the factors controlling polymorphism. All this stems from a delicate balance that the intermolecular D3 and solvent Floris, Tomasi and Pascual–Ahuir corrections provide. Combined optimised treatments should yield fully quantitative approaches in the future.     

ProSight Annotator: Complete control and customization of protein entries in UniProt XML files

The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms. Conversely, searching for a specific proteoform becomes a computationally difficult task for heavily modified proteins. Both situations require updates to the database through user-annotated entries. Unfortunately, manually creating properly formatted UniProt Extensible Markup Language (XML) files is tedious and prone to errors. ProSight Annotator solves these issues by providing a graphical interface for adding user-defined features to UniProt-formatted XML files for better informed proteoform searches. It can be downloaded from http://prosightannotator.northwestern.edu .     

MEGF10 is a mammalian ortholog of CED-1 that interacts with clathrin assembly protein complex 2 medium chain and induces large vacuole formation

The mechanisms underlying the engulfment of apoptotic corpses, which is involved in development, cellular homeostasis, and autoimmunity, remain largely unknown in mammals. MEGF10 is a mammalian ortholog of nematode CED-1, a transmembrane protein involved in engulfment of apoptotic corpses. MEGF10-expressing cells display an irregular, mosaic-like pattern of MEGF10, causing cells to tightly adhere to coated glass dishes. This restricted cell motility caused cells to adopt a flat appearance. In the present study, we observed that these cells formed unusually large vacuoles, the formation of which we linked to the cytoplasmic domain of MEGF10. While investigating the signaling pathway and trafficking of MEGF10, we identified an interaction between MEGF10 and clathrin assembly protein complex 2 medium chain (AP50), a component of clathrin-coated pits. In cells co-expressing MEGF10 and AP50, MEGF10 and AP50 colocalized and mirrored the adhesion pattern of MEGF10. LC-MS/MS and immunoblot analyses revealed that the MEGF10 associated with AP2 alpha and beta subunits in addition to associating with AP50 and beta-actin, and that MEGF10 was ubiquitinated and tyrosine phosphorylated. Moreover, we observed that MEGF10 mRNA expression is primarily restricted to the brain, with robust expression in the stellate cells of the cerebellum. Elucidating the trafficking and regulatory machinery of MEGF10 will guide us in having a deeper understanding of the mechanisms involved in clearing apoptotic cells.     

Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.     

Toxoplasma gondii: enzyme-linked immunosorbent assay using different fragments of recombinant microneme protein 1 (MIC1) for detection of immunoglobulin G antibodies

Three different fragments of microneme 1 protein termed, r-MIC1ex2, r-MIC1ex34 and r-MIC1 of Toxoplasma gondii, were expressed in Escherichia coli as fusion proteins containing six histidyl residues at N- and C-terminal. After purification by metal affinity chromatography, these recombinant proteins were tested for their usefulness as antigens in an enzyme-linked immunosorbent assay for the detection of immunoglobulin G. Ninety-eight sera from patients with different stages of invasion and 24 sera from seronegative patients were examined. There was no significant difference observed in the antigenicity for human serum samples from patients with acute toxoplasmosis between three recombinant types of MIC1 antigen (96.1% for r-MIC1ex2 antigen and 100% for both r-MIC1ex34 and r-MIC1 proteins). Sera from chronic infections (with low titers of IgG antibody) showed significant lower sensitivity, especially for r-MIC1ex34 and r-MIC1 antigens (75%, 52.7% and 36.1% for r-MIC1ex2, r-MIC1ex34 and r-MIC1, respectively). These results indicate that the strongest antigenic region of the MIC1 is encoding by the second exon of mic1 gene. When r-MIC1ex2 (N-terminal fragment of protein) was combined with MAG1 (matrix antigen) and MIC3 (microneme 3 protein), the sensitivity increased to 88.9%. This result was comparable to an ELISA using a Toxoplasma lysate antigen (TLA) and two combinations of recombinant antigens: M1 (GRA1+GRA7+SAG1) and M2 (P35+SAG2+GRA6) with the sensitivity for serum samples tested 94.4%, 88.9% and 94.4%, respectively.     

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

Pattern formation on the combs of honeybees: increasing fitness by coupling self-organization with templates

Biological patterns are often constructed via a combination of mechanisms including self-organization, templates and recipes. Our understanding of self-organization is becoming increasingly clear, yet how multiple mechanisms work together and what selective advantage they confer over simpler mechanisms is poorly understood. Honeybee (Apis mellifera) combs exhibit a pattern of brood at the bottom, pollen in a band next to it and honey at the top. This study constructs an agent-based model, derived from experimental studies, to determine both how self-organization interacts with two templates and to elucidate a selective basis for the use of multiple mechanisms. The vertical pattern of honey and brood is shown to be dependent on a gravity-based template, while the pollen band is shown to form via the interaction of a queen-based template and self-organization. The study suggests that the selective basis for this complex mechanism may be that colonies have higher growth rates when multiple mechanisms are used as opposed to self-organization alone. As self-organization is used in many contexts in which the addition of supplemental mechanisms could be advantageous, this result may be of general significance to many biological systems.     

The mechanism of amyloid-fibril formation by stefin B: temperature and protein concentration dependence of the rates

Cystatins, a family of structurally related cysteine proteinase inhibitors, have proved to be useful model system to study amyloidogenesis. We have extended previous studies of the kinetics of amyloid-fibril formation by human stefin B (cystatin B) and some of its mutants, and proposed an improved model for the reaction. Overall, the observed kinetics follow the nucleation and growth behavior observed for many other amyloidogenic proteins. The minimal kinetic scheme that best fits measurements of changes in CD and thioflavin T fluorescence as a function of protein concentration and temperature includes nucleation (modeled as N(I) irreversible transitions with equivalent rates (k(I)), which fitted with N(I) = 64), fibril growth and nonproductive oligomerization, best explained by an off-pathway state with a rate-limiting escape rate. Three energies of activation were derived from global fitting to the minimal kinetic scheme, and independently through the fitting of the individual component rates. Nucleation was found to be a first-order process within an oligomeric species with an enthalpy of activation of 55 +/- 4 kcal mol(-1). Fibril growth was a second-order process with an enthalpy of activation (27 +/- 5 kcal mol(-1)), which is indistinguishable from that of tetramer formation by cystatins, which involves limited conformational changes including proline trans to cis isomerization. The highest enthalpy of activation (95 +/- 5 kcal mol(-1) at 35 degrees C), characteristic of a substantial degree of unfolding as observed prior to domain-swapping reactions, equated with the escape rate of the off-pathway oligomeric state.     

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: Spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.