Native Stoichiometry Research Articles

Huntingtin S421 phosphorylation increases kinesin and dynein engagement on early endosomes and lysosomes

Huntingtin (HTT) is a scaffolding protein that recruits motor proteins to vesicular cargoes, enabling it to regulate kinesin-1, dynein, and myosin-VI-dependent transport. To maintain the native stoichiometry of HTT with its interacting partners, we used CRISPR/Cas9 to induce a phosphomimetic mutation of the endogenous HTT at S421 (HTT-S421D). Using single-particle tracking, optical tweezers, and immunofluorescence, we examined the effects of this mutation on the motility of early endosomes and lysosomes. In HTT-S421D cells, lysosomes exhibit longer displacements and higher processive fractions compared with wild-type (HTT-WT) cells. Kinesins and dyneins exert greater forces on early endosomes and lysosomes in cells expressing HTT-S421D. In addition, endosomes bind to microtubules faster and are more resistant to detachment under load. The recruitment of kinesins and dyneins to microtubules is enhanced in HTT-S421D cells. In contrast, overexpression of HTT had variable effects on the processivity, displacement, and directional bias of both early endosomes and lysosomes. These data indicate that phosphorylation of the endogenous HTT causes early endosomes and lysosomes to move longer distances and more processively by recruiting and activating both kinesin-1 and dynein.

Digital nanoreactors to control absolute stoichiometry and spatiotemporal behavior of DNA receptors within lipid bilayers

Interactions between membrane proteins are essential for cell survival but are often poorly understood. Even the biologically functional ratio of components within a multi-subunit membrane complex—the native stoichiometry—is difficult to establish. Here we demonstrate digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors (modelling membrane proteins). Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence, which in principle is generalizable to any ratio. Similar single-molecule-in-bulk experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

Xenopus Oocytes as a Powerful Cellular Model to Study Foreign Fully-Processed Membrane Proteins.

The use of Xenopus oocytes in electrophysiological and biophysical research constitutes a long and successful story, providing major advances to the knowledge of the function and modulation of membrane proteins, mostly receptors, ion channels, and transporters. Earlier reports showed that these cells are capable of correctly expressing heterologous proteins after injecting the corresponding mRNA or cDNA. More recently, the Xenopus oocyte has become an outstanding host–cell model to carry out detailed studies on the function of fully-processed foreign membrane proteins after their microtransplantation to the oocyte. This review focused on the latter overall process of transplanting foreign membrane proteins to the oocyte after injecting plasma membranes or purified and reconstituted proteins. This experimental approach allows for the study of both the function of mature proteins, with their native stoichiometry and post-translational modifications, and their putative modulation by surrounding lipids, mostly when the protein is purified and reconstituted in lipid matrices of defined composition. Remarkably, this methodology enables functional microtransplantation to the oocyte of membrane receptors, ion channels, and transporters from different sources including human post-mortem tissue banks. Despite the large progress achieved over the last decades on the structure, function, and modulation of neuroreceptors and ion channels in healthy and pathological tissues, many unanswered questions remain and, most likely, Xenopus oocytes will continue to help provide valuable responses.

A Routine 'Top-Down' Approach to Analysis of the Human Serum Proteome.

Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3–10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7–20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes.

Exploring alternate states and oligomerization preferences of coiled-coils by de novo structure modeling.

Homomeric coiled-coils can self-assemble into a wide range of structural states with different helix topologies and oligomeric states. In this study, we have combined de novo structure modeling with stability calculations to simultaneously predict structure and oligomeric states of homomeric coiled-coils. For dimers an asymmetric modeling protocol was developed. Modeling without symmetry constraints showed that backbone asymmetry is important for the formation of parallel dimeric coiled-coils. Collectively, our results demonstrate that high-resolution structure of coiled-coils, as well as parallel and antiparallel orientations of dimers and tetramers, can be accurately predicted from sequence. De novo modeling was also used to generate models of competing oligomeric states, which were used to compare stabilities and thus predict the native stoichiometry from sequence. In a benchmark set of 33 coiled-coil sequences, forming dimers to pentamers, up to 70% of the oligomeric states could be correctly predicted. The calculations demonstrated that the free energy of helix folding could be an important factor for determining stability and oligomeric state of homomeric coiled-coils. The computational methods developed here should be broadly applicable to studies of sequence-structure relationships in coiled-coils and the design of higher order assemblies with improved oligomerization specificity.

Homodimers of the Claudin‐2 Polymerize to Tight Junctions of Canine Kidney Epithelial Cells

Claudin‐2 (CLDN‐2) is a member of a family of 27 tight junction transmembrane proteins. Claudins regulate paracellular ion transport in mammalian epithelial cells by forming either a barrier or a size‐ and charge‐selective pore. CLDN‐2 forms a high‐conductance cation pore, and controls sodium ion permeability. Little is known about the structural basis for ion selectivity of claudins. Their molecular organization into multiprotein complexes at tight junctions is also poorly understood. Recent biochemical studies have suggested that CLDN‐2 can form stable dimers in Madin‐Darby canine kidney (MDCK) II cells, while our mutagenesis studies indicate that CLDN‐2 pores exist as high order multimers. In the current work, we overexpressed tandem homodimers of CLDN‐2 in high‐resistance MDCK I TetOff cells. By Western blotting, cells exhibited inducible expression of CLDN‐2 dimers (~33 kDa) in the absence of doxycycline (−dox), which was almost completely suppressed by doxycycline. By immunofluorescence, the dimeric species of CLDN‐2 were colocalized with ZO‐1 at the apical junctional complex, with some additional intracellular staining at perinuclear area. The transepithelial resistance of cells overexpressing dimers of CLDN‐2 (−dox) was 1.5–2 times lower than in the presence of doxycycline. Our results indicate that tandem dimers of CLDN‐2 polymerize to tight junctions and function normally as paracellular pores suggesting that the native stoichiometry of CLDN‐2 is a multiple of two subunits. Our study may help to understand how claudins are organized into oligomers at tight junctions. This study is supported by NIH grant U01‐GM094627

Structural study on the architecture of the bacterial ATP synthase F o motor

We purified the F(o) complex from the Ilyobacter tartaricus Na(+)-translocating F(1)F(o)-ATP synthase and performed a biochemical and structural study. Laser-induced liquid bead ion desorption MS analysis demonstrates that all three subunits of the isolated F(o) complex were present and in native stoichiometry (ab(2)c(11)). Cryoelectron microscopy of 2D crystals yielded a projection map at a resolution of 7.0 Å showing electron densities from the c(11) rotor ring and up to seven adjacent helices. A bundle of four helices belongs to the stator a-subunit and is in contact with c(11). A fifth helix adjacent to the four-helix bundle interacts very closely with a c-subunit helix, which slightly shifts its position toward the ring center. Atomic force microscopy confirms the presence of the F(o) stator, and a height profile reveals that it protrudes less from the membrane than c(11). The data limit the dimensions of the subunit a/c-ring interface: Three helices from the stator region are in contact with three c(11) helices. The location and distances of the stator helices impose spatial restrictions on the bacterial F(o) complex.

Native TRPC7 Channel Activation by an Inositol Trisphosphate Receptor-dependent Mechanism

In DT40 B lymphocytes, Canonical Transient Receptor Potential 7 (TRPC7) functions as a diacylglycerol-activated non-selective cation channel. However, previous work indicated that the non-store-operated Ca2+ entry in this cell type depends upon inositol trisphosphate receptors (IP3R). With the cell-attached configuration oleyl-acetyl-glycerol (OAG) induced single channel activity (75 pS) that was not observed in TRPC7-/- cells but was rescued by expression of TRPC7 under conditions expected to produce relatively low levels of expression ((LowT7)TRPC7-/-). A DT40 cell line lacking IP3R(IP3R-/- cells) showed no OAG-induced single channel activity, but this activity was rescued by transient expression of an IP3R((IP3R)IP3R-/-). Single channel properties in (LowT7)TRPC7-/- or (IP3R)IP3R-/- DT40 cells were indistinguishable from one another and from wild-type cells. Thus, TRPC7 forms, or is part of, the channel underlying endogenous diacylglycerol-activated currents in DT40 B lymphocytes, and this activity of native TRPC7 requires IP3R. However, with conditions expected to produce greater expression levels, TRPC7 functioned independently of the presence of IP3R. This finding may serve to resolve previously conflicting reports from expression studies of TRPC channels.

Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.