Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.     

X-ray crystallographic structures of the Escherichia coli periplasmic protein FhuD bound to hydroxamate-type siderophores and the antibiotic albomycin.

Siderophore-binding proteins play an essential role in the uptake of iron in many Gram-positive and Gram-negative bacteria. FhuD is an ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of hydroxamate-type siderophores in Escherichia coli. Structures of FhuD complexed with the antibiotic albomycin, the fungal siderophore coprogen and the drug Desferal have been determined at high resolution by x-ray crystallography. FhuD has an unusual bilobal structure for a periplasmic ligand binding protein, with two mixed beta/alpha domains connected by a long alpha-helix. The binding site for hydroxamate-type ligands is composed of a shallow pocket that lies between these two domains. Recognition of siderophores primarily occurs through interactions between the iron-hydroxamate centers of each siderophore and the side chains of several key residues in the binding pocket. Rearrangements of side chains within the binding pocket accommodate the unique structural features of each siderophore. The backbones of the siderophores are not involved in any direct interactions with the protein, demonstrating how siderophores with considerable chemical and structural diversity can be bound by FhuD. For albomycin, which consists of an antibiotic group attached to a hydroxamate siderophore, electron density for the antibiotic portion was not observed. Therefore, this study provides a basis for the rational design of novel bacteriostatic agents, in the form of siderophore-antibiotic conjugates that can act as "Trojan horses," using the hydroxamate-type siderophore uptake system to actively deliver antibiotics directly into targeted pathogens.     

New species of the Hygrobates salamandrarum-group (Acari, Hydrachnidia, Hygrobatidae) from Southeast Asia

Two new water mite species of the genus Hygrobates Koch, 1837 (Acari, Hydrachnidia, Hygrobatidae) were found to live parasitic on newts of the genus Paramesotriton Chang, 1935 (Amphibia, Caudata, Salamandridae) from Vietnam and Laos: Hygrobates forcipifer sp. nov. and H. ancistrophorus sp. nov. The H. salamandrarum-group is defined, that now includes three species from Southeast Asia. Males and females of both new species are described, as well as larvae and deutonymphs of the Vietnamese species. These data provide the first record of males, nymphs and larvae of the species-group. The systematic position of the group, as well as the parasite-host association and the lifecycle of the species are discussed. Furthermore, the character states of the striking mouthparts, particularly modified as an adaptation for penetrating the amphibian skin, the genital skeleton and the larval morphology are examined.     

Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins.

Mdj1p, a DnaJ homolog in the mitochondria of Saccharomyces cerevisiae, is involved in the folding of proteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1 was constructed. Mutations in the various functional domains led to a partial loss of Mdj1p function. The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregation in isolated mitochondria. The mitochondrially encoded var1 protein showed enhanced aggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordance with the lack of requirement of Mdj1p in the mt-Hsp70-mediated protein import into mitochondria. On the other hand, precursor proteins in transit which had further entered the matrix were found in a complex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone for mitochondrially synthesized polypeptide chains emerging from the ribosome and for translocating proteins at a late import step.     

Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA

Protein arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). Three forms of methylarginine have been identified in eukaryotes: monomethylarginine (l-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. l-NMMA and ADMA, but not SDMA, are competitive inhibitors of all nitric oxide synthase isoforms. SDMA is eliminated almost entirely by renal excretion, whereas l-NMMA and ADMA are further metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To explore the interplay between methylarginine synthesis and degradation in vivo, we determined PRMT expression and DDAH activity in mouse lung, heart, liver, and kidney homogenates. In addition, we employed HPLC-based quantification of protein-incorporated and free methylarginine, combined with immunoblotting for the assessment of tissue-specific patterns of arginine methylation. The salient findings of the present investigation can be summarized as follows: 1) pulmonary expression of type I PRMTs was correlated with enhanced protein arginine methylation; 2) pulmonary ADMA degradation was undertaken by DDAH1; 3) bronchoalveolar lavage fluid and serum exhibited almost identical ADMA/SDMA ratios, and 4) kidney and liver provide complementary routes for clearance and metabolic conversion of circulating ADMA. Together, these observations suggest that methylarginine metabolism by the pulmonary system significantly contributes to circulating ADMA and SDMA levels.     

Phytochrome and UV signal transduction pathways

The phytochromes are the best studied plant photoreceptors, controlling a wide variety of responses at both whole plant and single cell levels. Three signal transduction pathways, dependent on cGMP and/or calcium, have been found to be utilized by phytochrome to control the expression of genes required for chloroplast development (e.g., CAB and FNR) and anthocyanin biosynthesis (e.g., CHS). In particular, cGMP is a second messenger positively regulating CHS gene expression whilst calcium and calmodulin act as negative regulators. In addition to phytochrome regulation of CHS we have begun to examine the signal transduction pathways utilized by UV photoreceptors. In contrast to phytochrome-mediated responses, results indicate a role for calcium and calmodulin as positive regulators of CHS gene expression in UV light.     

SAR of N-phenyl piperidine based oral integrin α5β1 antagonists

Recently, a new class of selective integrin α5β1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives are described.     

Distinct steps in the import of ADP/ATP carrier into mitochondria

Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria.