/pdf/recent-advances-in-the-biochemistry-of-polyamines-in-382j7cp9ea.pdf

Recent advances in the biochemistry of polyamines in eukaryotes.

Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character No highly conserved 'homology blocks' are found in either class of topogenic sequence Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1989.tb14679.x

Domain structure of mitochondrial and chloroplast targeting peptides.

In this critical review, I would like to provide a brief outline of the morphology, biochemical composition, distribution, and functions of peroxisomes. The induction of peroxisome proliferation and peroxisome-associated enzymes in the rodent liver by two classes of chemicals (hypolipidemic drugs and the industrial plasticizers) will be considered. The role of peroxisomes in lipid metabolism will be discussed. Carcinogenicity studies in rats and mice with these peroxisome proliferators will be evaluated critically. Careful consideration will be given to the hypothesis that "potent hepatic peroxisome proliferators as a class are carcinogenic." The possible mechanism(s) by which peroxisome proliferators induce liver tumors will be outlined. Particular attention will be paid to the possible role of peroxisome proliferation-mediated radical toxicity and generation of endogenous initiators of carcinogenesis.

Carcinogenesis by Hepatic Peroxisome Proliferators: Evaluation of the Risk of Hypolipidemic Drugs and Industrial Plasticizers to Humans

Altered Constitutive Expression of Fatty Acid-metabolizing Enzymes in Mice Lacking the Peroxisome Proliferator-activated Receptor α (PPARα)

Because of the high degree of organizational complexity of eucaryotic cells, it is clear that the implementation of their genetic programs must, in many cases, involve a complex sequence of cotranslational and posttranslational events that are necessary to transfer polypeptides from their sites of synthesis to their sites of function. It is difficult to envisage the existence of a single general mechanism that would ensure that all polypeptides released from ribosomes attain their correct subcellular destination . This is because there are, in a eucaryotic cell, at least as many possible destinations for a newly synthesized polypeptide as there are different compartments and membrane systems. Moreover, it is not likely that completed and fully folded polypeptides, with their charged and polar residues exposed to the aqueous environment, could freely traverse hydrophobic barriers constituted by phospholipid bilayers, which are the universal feature of all cell membranes (208). Instead, it may be expected that special mechanisms have evolved that direct polypeptides to specific membranes and, when necessary, assist them in their passage across the hydrophobic barriers . These mechanisms must involve specific receptors for structural features ofthe polypeptides and may entail conformational changes or even extensive structural modifications of the polypeptide, as well as the expenditure of energy . In this paper we consider mechanisms for the transfer of newly synthesized polypeptides to their sites of function in different subcellular membranes and organelles, and discuss models in which specific features ofthe polypeptides serve as signals to direct them along selected subcellular pathways to their final destination . These signals may act during translation or after synthesis of the polypeptide is completed and may or may not be removed from the initial product of translation . Transient or permanent signals within polypeptides destined to membranes would also account for the final characteristic orientation of membrane proteins with respect to the phospholipid bilayer . Several other reviews discussing various aspects of this subject have recently appeared (15, 44, 53, 117a, 127, 243a) .

https://rupress.org/jcb/article-pdf/92/1/1/1075487/1.pdf

Mechanisms for the incorporation of proteins in membranes and organelles.

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.

Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver.

Ornithine carbamoyltransferase of rat liver mitochondrial matrix (subunit Mr= 36000) is synthesi/ed extramitochondrially as a larger precursor (subunit Mr= 39400) which is transported into mitochondria, in association with post-translational proteolytic processing. Rat liver mitochondria convert the precursor to the mature enzyme as well as to a 37000-Mr product. a possible intermediate of the processing [Mori, M., Miura, S., Tatibana. M., and Cohen, P. P. (1980) Proc. Natl Acad. Sci. USA, 77, 7044-7048]. A protease responsible for the conversion of the precursor to the 37000-Mr product was purified 140-fold from the matrix fraction of rat liver mitochondria. The protease had an estimated Mr of 108000 and an apparent pl of 5.5. Mature ornithine carbamoyltransferase (0.5 μg) did not inhibit the cleavage of the precursor by the protease and presumably the latter cleaves a specific site on the extrapeptide of the carbamoyltransferase precursor. The protease was inhibited by metal-chelating reagents such as EDTA, o-phenanthroline and zincon and by a high concentration (1 mM) of leupeptin. It did not cleave several of the protein and peptide substrates tested including the precursor of mitochondrial carbamoyl-phosphate synthetase I. Apparently the same protease activity is widely distributed among mitochondria of rat kidney, spleen, heart and ascites tumor cells, all of which lack ornithine carbamoyltransferase. A possible physiological role of the protease in the processing of the mitochondria1 protein precursors is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1982.tb06487.x

A Mitochondria1 Protease that Cleaves the Precursor of Ornithine Carbamoyltransferase

Total RNA or poly(A)+ RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.

Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver.

The sorting of homologous proteins between two separate intracellular organelles is a major unsolved problem 3-Oxoacyl-CoA thiolase is localized in mitochondria and peroxisomes, and provides a good system for the study on the problem Unlike most mitochondrial matrix proteins, mitochondrial 3-oxoacyl-CoA thiolase in rats is synthesized with no transient presequence and possess information for mitochondrial targeting and import in the mature protein Two overlapping cDNA clones contained an open reading frame encoding a polypeptide of 397 amino acid residues (predicted Mr = 41,868), a 5' untranslated sequence of 164 bp, a 3' untranslated sequence of 264 bp and a poly(A) tract The amino acid sequence of the mitochondrial thiolase is 37% identical with that of the mature portion of rat peroxisomal 3-oxoacyl-CoA thiolase precursor These results suggest that the two thiolases have a common origin and obtained information for targeting to respective organelles during evolution Two portions in the mitochondrial thiolase that may serve as a mitochondrial targeting signal are presented

cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

Messenger RNA of rat ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, was enriched by immunoprecipitation of rat liver free polysomes, and recombinant plasmids were prepared from the enriched mRNA by a vector-primer method. The cDNA clones for ornithine carbamoyltransferase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pOTC-1, contained a 1.6-kilobase insert and hybridized to a mRNA of approximately equal to 1.8 kilobases in rat liver. The cDNA clone was subjected to nucleotide sequence analysis. The deduced amino acid sequence indicates that the ornithine carbamoyltransferase precursor consists of the mature enzyme of 322 amino acid residues and an NH2-terminal peptide extension (presequence) of 32 amino acid residues. The presequence contains 8 basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The amino acid sequence of the rat ornithine carbamoyltransferase was compared with the recently reported sequence of the human enzyme [Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W. & Rosenberg, L. E. (1984) Science 224, 1068-1074]. The sequences of the mature enzyme portion are 93% identical, whereas those of the presequences are 69% identical. There are two highly conserved segments in the presequences of the rat and human enzymes. One of the two conserved segments is significantly similar to a segment of the presequence of yeast mitochondrial elongation factor EF-Tu. These results suggest that the homologous segments are important for the proteins that are synthesized in the cytosol to be transported into the mitochondrial matrix.

Masataka Mori

Papers

Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver.

A Mitochondria1 Protease that Cleaves the Precursor of Ornithine Carbamoyltransferase

Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver.

cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme.

Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.