Neurofilament Protein Phosphorylation in Spinal Cord of Experimentally Diabetic Rats

This study was designed to determine if the known decrease in slow axonal transport of proteins in the sciatic nerve of experimentally diabetic rats is related to altered phosphorylation of neurofilament proteins (NFPs). Rats were rendered diabetic with 50 mg/kg of streptozotocin, i.p. At 3 and 6 weeks later, NFPs were prepared from spinal cord. The in vivo phosphorylation state of NFPs was examined by using phosphate-dependent (RT97) and -independent (RMd09) antibodies against high-molecular-mass NFPs on Western blots. Neurofilament-associated kinase activity was also measured in vitro by incubation of NFPs with [32P]ATP. Phosphorylation of all three NFPs (high, medium, and low molecular mass) occurred, as confirmed by gel electrophoresis and autoradiography. At 30 min of incubation, protein-bound radioactivity in NFPs from diabetic animals was reduced to 86.7 +/- 3.4 and 54.3 +/- 19.6% of that in nondiabetic animals at 3 and 6 weeks of diabetes, respectively (p less than 0.001 and p less than 0.05, respectively). NFPs were also incubated with acid phosphatase and rephosphorylated. Results showed that the increased in vivo phosphorylation contributed to the decreased in vitro phosphorylation. Extraction of protein kinases and addition back to the NFPs revealed, in addition, a reduced activity in the diabetic animals of the protein kinases measured in vitro.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Hyperthermia Induces the Synthesis of a Heat Shock Protein by Polysomes Isolated from the Fetal and Neonatal Mammalian Brain

Abstract: Analysis of the cell-free translation products of polysomes isolated from fetal brain and other organs indicates that elevation of maternal body temperature induces the synthesis of a heat shock protein of molecular weight 74,000 (74K). The newborn mammal is particularly sensitive to induction of the 74K protein. As early postnatal development proceeds, higher body temperatures are required to induce synthesis of the 74K heat shock protein.     

Validation of the first step of the heuristic refinement method for the derivation of solution structures of proteins from NMR data

A new method for the analysis of NMR data in terms of the solution structure of proteins has been developed. The method consists of two steps: first a systematic search of the conformational space to define the region allowed by the initial set of experimental constraints, and second, the narrowing of this region by the introduction of additional constraints and optional refinement procedures. The search of the conformational space is guided by heuristics to make it computationally feasible. The method is therefore called the heuristic refinement method and is coded in an expert system called PROTEAN. The paper describes the validation of the first step of the method using an artificial NMR data set generated from the known crystal structure of sperm whale carbon monoxymyoglobin. It is shown that the initial search procedure yields a low-resolution structure of the myoglobin molecule, accurately reproducing its main topological features, and that the precision of the structure depends on the quality of the initial data set.