Idealized Models of Protofilaments of Human Islet Amyloid Polypeptide

Fibrils formed by assembly of human islet amyloid polypeptide (hIAPP) are found in most patients with type II diabetes. Structurally, these fibrils are composed of multiple protofilaments and are characterized by extended beta sheets, variable helical twists, and different morphologies. We have previously derived models for the hIAPP protofilament using simulations constrained by data from EPR spectroscopy. In the current work, these models were used as a basis for generating idealized hIAPP protofilaments with symmetrical geometrical properties using a new algorithm, MFIBRIL. We show good agreement of the idealized protofilaments with experimental data for amino acid side chain orientations and geometrical features including the inter-β sheet distance and the protofilament radius. These idealized protofilaments can be used in MFIBRIL to generate fibril models that may be experimentally testable at the molecular level. MFIBRIL can also be used for building structures of any repetitive molecular assembly starting with a single building block obtained from any source

Secondary and Tertiary Structure of Bacteriorhodopsin in the SDS Denatured State

We characterized the structure of partially unfolded bacteriorhodopsin in sodium dodecyl sulfate (SDS) micelles and compared it with its <i>in vitro</i> refolded structure after reconstitution with dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)­dimethylammonio]-1-propanesulfonate (DMPC/CHAPS). Intrahelical and interhelical distances were mapped in the protein using strategically located spin-label pairs at helical ends, assayed by pulsed electron paramagnetic resonance spectroscopy (double electron–electron spin resonance, DEER). We find that in SDS the intrahelical end-to-end distances exhibit broad distributions, suggesting a heterogeneous ensemble of conformations with differing secondary structures. Nevertheless, a majority of the denatured population retains end-to-end distances similar to those in the native state. In contrast, the observed greatly increased interhelical distances, in addition to their very broad distributions, suggest that in the SDS micelles very little of the native tertiary structure remains

Hydration Dynamics of a Peripheral Membrane Protein

Water dynamics in the hydration shell of the peripheral membrane protein annexin B12 were studied using MD simulations and Overhauser DNP-enhanced NMR. We show that retardation of water motions near phospholipid bilayers is extended by the presence of a membrane-bound protein, up to around 10 Å above that protein. Near the membrane surface, electrostatic interactions with the lipid head groups strongly slow down water dynamics, whereas protein-induced water retardation is weaker and dominates only at distances beyond 10 Å from the membrane surface. The results can be understood from a simple model based on additive contributions from the membrane and the protein to the activation free energy barriers of water diffusion next to the biomolecular surfaces. Furthermore, analysis of the intermolecular vibrations of the water network reveals that retarded water motions near the membrane shift the vibrational modes to higher frequencies, which we used to identify an entropy gradient from the membrane surface toward the bulk water. Our results have implications for processes that take place at lipid membrane surfaces, including molecular recognition, binding, and protein–protein interactions

Nonaggregated α‑Synuclein Influences SNARE-Dependent Vesicle Docking via Membrane Binding

α-Synuclein (α-Syn), a major component of Lewy body that is considered as the hallmark of Parkinson’s disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn buildup inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of nonaggregated α-Syn on SNARE-dependent liposome fusion using fluorescence methods. In ensemble in vitro assays, α-Syn reduces lipid mixing mediated by SNAREs. Furthermore, with the more advanced single-vesicle assay that can distinguish vesicle docking from fusion, we found that α-Syn specifically inhibits vesicle docking, without interfering with the fusion. The inhibition in vesicle docking requires α-Syn binding to acidic lipid containing membranes. Thus, these results imply the existence of at least two mechanisms of inhibition of SNARE-dependent membrane fusion: at high concentrations, nonaggregated α-Syn inhibits docking by binding acidic lipids but not v-SNARE; on the other hand, at much lower concentrations, large α-Syn oligomers inhibit via a mechanism that requires v-SNARE interaction [Choi et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (10), 4087−4092]

Semisynthetic, Site-Specific Ubiquitin Modification of α-Synuclein Reveals Differential Effects on Aggregation

The process of neurodegeneration in Parkinson’s Disease is intimately associated with the aggregation of the protein α-synuclein into toxic oligomers and fibrils. Interestingly, many of these protein aggregates are found to be post-translationally modified by ubiquitin at several different lysine residues. However, the inability to generate homogeneously ubiquitin modified α-synuclein at each site has prevented the understanding of the specific biochemical consequences. We have used protein semisynthesis to generate nine site-specifically ubiquitin modified α-synuclein derivatives and have demonstrated that different ubiquitination sites have differential effects on α-synuclein aggregation

The S100A10 Subunit of the Annexin A2 Heterotetramer Facilitates L2-Mediated Human Papillomavirus Infection

<div><p>Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108–120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.</p> </div

Mutations in HPV16 L2_108–111 reduce PsV binding to A2t and PsV infectivity.

<p>(A) ELISA plate wells were coated with 500 ng of A2t prior to overnight incubation with 400 ng HPV16 PsV or HPV16 L1–L2(GGDD) mutant PsV and subsequently incubated with mouse anti-L1 H16.V5 or goat anti-SLPI antibodies. Anti-mouse and anti-goat HRP-conjugated secondary antibodies were added prior to the substrate. In control experiments, no ligands were used. The graph represents the mean absorbance measured at 490 nm ± SD (***<i>P</i><0.001 as determined by a two-tailed, unpaired t-test between WT and mutant PsV). (B) HaCaT cells were infected with wild type (WT) or mutant (L2_108–111 LVEE to GGDD) HPV16 pseudovirions containing a GFP plasmid. Infectivity was scored at 48 h post infection by enumerating GFP-positive cells by flow cytometry. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the WT PsV group ± SD are presented of two combined independent experiments. Inset shows the L1 band of a coomassie blue stained SDS-PAGE gel loaded with an equivalent amount of WT and mutant PsV used in the infectivity assays. (***<i>P</i><0.001 as determined by a two-tailed, unpaired t-test between WT and mutant PsV group).</p

Surface expression of A2t on HaCaT and HeLa human epithelial cell lines. (

<p>A) HaCaT and HeLa cells were incubated with an anti- S100A10 antibody, then incubated with fluorophore-conjugated secondary antibodies, and mounted with DAPI containing media. For control staining, cells were either stained with a mouse or rabbit IgG isotype control followed by secondary antibody staining. Images were acquired using an upright confocal fluorescent microscope. (B) HeLa and HaCaT cells were incubated with PBS supplemented with Ca²⁺ or PBS with increasing concentration of EDTA for 45 min. The supernatants were collected and the presence of ANXA2 and S100A10 were analyzed via Western blot.</p

shRNA knockdown of A2t reduces internalization of HPV16 L1L2 VLP and infectivity of HPV16 PsV.

<p>(A) HPV16 L1 VLP were fluorescently labeled with CFDA-SE, and the percent of infected cells was measured as the percent that were CFDA-SE positive after exposure to VLP for 1 hour as measured by FACS. To control for free label false positives, cells were treated with VLP pre-incubated with a neutralizing anti-L1 antibody (H16.V5). An identical experiment was performed using HPV16 L1L2 VLP. Both histograms are representative examples of two experiments done in triplicate. (B) HeLa cells were left untreated or transduced with a doxycycline inducible pTRIPZ Tet-On lentiviral vector containing an shRNA against ANXA2. Single cell clones treated with or without doxycycline were incubated with labeled HPV16 VLP for 3 hours at 37° and assessed by FACS. The mean percentage of uptake normalized to the wild type group ± SEM of three independent experiments is presented. (C) Protein was collected from cell populations used in the internalization assay for analysis of ANXA2 and S100A10 via Western blot. GAPDH served as a loading control. (D) mRNA was collected from cell populations in the uptake assay for quantitative RT-PCR analysis of ANXA2 and S100A10 expression. The mRNA expression levels were normalized to GAPDH and the graph is a representative example of an experiment performed in triplicate ± SD. (E) Wildtype HeLa cells or HeLa cells stably transduced with a doxycycline inducible lentiviral vector containing shRNA against ANXA2 or control non-target lentiviral vector were infected with GFP plasmid containing HPV16 pseudovirus. Infection was scored 48 h later by enumeration of GFP-positive cells by flow cytometry. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the no doxycycline treated groups ± SD are presented. (*<i>P</i><0.05 and **<i>P</i><0.01 as determined by a two-tailed, unpaired t-test, as compared to the no doxycycline-treated groups). Figure is representative of two independent experiments. (F) Protein was collected from cell populations used in the infection assay for analysis of ANXA2 and S100A10 via Western blot. GAPDH served as a loading control.</p

HPV16 PsV infection decreases following SLPI treatment or anti-annexin A2 antibody inhibition of A2t.

<p>HaCaT cells were infected with HPV16 pseudovirions containing a GFP plasmid. Infectivity was scored at 48 h post infection by enumerating GFP-positive cells by flow cytometry. (A) Cells were preincubated with increasing amounts of SLPI or BSA for one hour at 4° prior to PsV infection. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the PsV only group ± SD are presented. (**<i>P</i><0.01 as determined by a two-tailed, unpaired t-test, as compared to the PsV only group). (B) Cells were incubated with increasing amounts of an anti-annexin A2 Ab or isotype control (mouse IgG1) for one hour prior to PsV infection The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the PsV only group ± SD are presented (**<i>P</i><0.01 as determined by a two-tailed, unpaired t-test, as compared to PsV only except where otherwise noted). Each graph is representative of at least two independent experiments.</p