The mitochondrial calcium uniporter is a multimer that can include a dominant‐negative pore‐forming subunit

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red‐sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino‐acid substitutions in the pore region and indeed MCUb does not form a calcium‐permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant‐negative effect, reducing the [Ca²⁺]_mt increases evoked by agonist stimulation. Accordingly, in vitro co‐expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant‐negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.     

Intramolecular Dynamics within the N-Cap-SH3-SH2 Regulatory Unit of the c-Abl Tyrosine Kinase Reveal Targeting to the Cellular Membrane

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl⁺ cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.     

Discovery of a Unique Clp Component,ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery

Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome.     

Influence of Dendritic Cells on Viral Pathogenicity

Although most viral infections cause minor, if any, symptoms, a certain number result in serious illness. Viral disease symptoms result both from direct viral replication within host cells and from indirect immunopathological consequences. Dendritic cells (DCs) are key determinants of viral disease outcome; they activate immune responses during viral infection and direct T cells toward distinct T helper type responses. Certain viruses are able to skew cytokine secretion by DCs inducing and/or downregulating the immune system with the aim of facilitating and prolonging release of progeny. Thus, the interaction of DCs with viruses most often results in the absence of disease or complete recovery when natural functions of DCs prevail, but may lead to chronic illness or death when these functions are outmanoeuvred by viruses in the exploitation of DCs.     

Generation in Human Plasma of Misfolded, Aggregation-Prone Electronegative Low Density Lipoprotein

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(−), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(−) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(−) during incubation of unprocessed human plasma at 37°C. In addition to a higher fraction of amyloidogenic LDL(−), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone.     

Characterization of dynorphin A-converting enzyme in human spinal cord. An endoprotease related to a distinct conversion pathway for the opioid heptadecapeptide?

A highly specific proteinase, converting dynorphin A (1-17) to enkephalins, was isolated from the human spinal cord and subjected to further characterization. The enzyme was found to be a thiol-dependent protein with a relative molecular mass of 50 kDa and a pH optimum between 5.0 and 5.5. This proteinase appears to exclusively convert dynorphin A (1-17) to Leu-enkephalin and its COOH-terminal extensions Leu-enkephalin-Arg6 (which was a major conversion product) and Leu-enkephalin-Arg6-Arg7 but not the other prodynorphin- or proenkephalin-derived peptides. This high specificity toward a single structure is suggested to be involved in a distinct processing pathway associated with the generation of the opioid peptides with selectivity for delta-opioid receptors.     

Seeing in the swamp: hydrogen sulfide inhibits eye metabolism and visual acuity in a sulfide-tolerant fish

In fish, vision may be impaired when eye tissue is in direct contact with environmental conditions that limit aerobic ATP production. We hypothesized that the visual acuity of fishes exposed to hydrogen sulfide (H₂S)-rich water would be altered owing to changes in cytochrome c oxidase (COX) activity. Using the H₂S-tolerant mangrove rivulus (Kryptolebias marmoratus), we showed that a 10 min exposure to greater than or equal to 200 µM of H₂S impaired visual acuity and COX activity in the eye. Visual acuity and COX activity were restored in fish allowed to recover in H₂S-free water for up to 1 h. Since K. marmoratus are found in mangrove pools with H₂S concentrations exceeding 1000 µM, visual impairment may impact predator avoidance, navigation and foraging behaviour in the wild.