A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate

Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates

Adjustable Ellipsoid Nanoparticles Assembled from Re-engineered Connectors of the Bacteriophage Phi29 DNA Packaging Motor

A 24 x 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the bacteriophage phi29 DNA packaging motor was constructed. Homogeneous nanoparticles were obtained with simple one-step purification. Electron microscopy and analytical ultracentrifugation were employed to elucidate the structure, shape, size, and mechanism of assembly. The formation of this structure was mediated and stabilized by N-terminal peptide extensions. Reversal of the 84-subunit ellipsoid nanoparticle to its dodecamer subunit was controlled by the cleavage of the extended N-terminal peptide with a protease. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.</p> <p>Methods</p> <p>Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.</p> <p>Results</p> <p>Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the <it>in vitro </it>data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation <it>in vivo</it>. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.</p> <p>Conclusions</p> <p>Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.</p

Electron microscopy and image analysis of the GroEL-like protein and its complexes with glutamine synthetase from pea leaves

The molecular structure of groEL-like protein from pea leaves has been studied by electron microscopy and image analysis of negatively stained particles. Over 1500 molecular projections were selected and classified by multivariate statistical analysis. It was shown that the molecule consists of 14 subunits arranged in two layers with 72 point group symmetry. Side view projections of the molecule show a four-striation appearance, which subdivides both layers of seven subunits into two halves; this may be explained by a two-domain structure of the subunits. The presence in protein preparations of projections corresponding to one layer of subunits or half-molecules is consistent with the molecular structure suggested. Electron microscopic evidence for a specific association of GroEL-like protein and octameric glutamine synthetase, which was co-purified with this protein, was obtained

SCANNING TUNNELING MICROSCOPY (STM) OF THE MO-FE PROTEIN-COMPONENT OF AZOTOBACTER-VINELANDII NITROGENASE - IDENTIFICATION OF THE SUBUNIT STRUCTURES

Müller A, Diemann E, Knuttel K, Branding A, Schneider K. SCANNING TUNNELING MICROSCOPY (STM) OF THE MO-FE PROTEIN-COMPONENT OF AZOTOBACTER-VINELANDII NITROGENASE - IDENTIFICATION OF THE SUBUNIT STRUCTURES. Naturwissenschaften. 1991;78(11):507-508

Three-dimensional Architecture of Hair-bundle Linkages Revealed by Electron-microscopic Tomography

The senses of hearing and balance rest upon mechanoelectrical transduction by the hair bundles of hair cells in the inner ear. Located at the apical cellular surface, each hair bundle comprises several tens of stereocilia and a single kinocilium that are interconnected by extracellular proteinaceous links. Using electron-microscopic tomography of bullfrog saccular sensory epithelia, we examined the three-dimensional structures of basal links, kinociliary links, and tip links. We observed significant differences in the appearances and dimensions of these three structures and found two distinct populations of tip links suggestive of the involvement of different proteins, splice variants, or protein–protein interactions. We noted auxiliary links connecting the upper portions of tip links to the taller stereocilia. Tip links and auxiliary links show a tendency to adopt a globular conformation when disconnected from the membrane surface