Cytoplasmic cleavage

Fujiwara, K., Pollard, T.D. (1976). Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow and mitotic spindle of human cells. J. Cell Biol. 71, 848-875. 

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

Fig. 2 RNAi inducers used in antiviral strategies. In general, RNAi is induced either by transfection of synthetic siRNAs into cells, or by stable or transient intracellular expression of double-stranded siRNA precursors (shRNA, e-shRNA, IhRNA, or pri-miRNAs). After transcription in the nucleus shRNAs, IhRNAs and e-shRNAs are exported to the cytoplasm and subsequently diced into mature siRNAs. Pri-miRNAs modified to encode antiviral siRNAs first undergo cleavage by Drosha before they are exported to the cytoplasm. Here the antiviral pre-miRNAs (also called shRNA-miRs) are processed by Dicer into the mature miRNAs. After loading of the antisense strand of the siRNAs/miRNAs into RISC, the complex will target and cleave viral transcripts bearing the complementary sequences...

Nonmuscle cells perform mechanical work, including self-propulsion, morphogenesis, cleavage, endocytosis, exocytosis, intracellular transport, and changing cell shape. These cellular functions are carried out by an extensive intracellular network of filamentous structures constimting the cytoskeleton. The cell cytoplasm is not a sac of fluid, as once thought. Essentially all eukaryotic cells contain three types of filamentous struc-mres actin filaments (7-9.5 nm in diameter also known as microfilaments), microtubules (25 nm), and intermediate filaments (10-12 nm). Each type of filament can be distinguished biochemically and by the electron microscope. 

Proteolytic cleavage has proven to be an efficient tool for exploring the structure and function of the Na,K-ATPase. Exposure and protection of bonds on the surface of the cytoplasmic protrusion provides unequivocal evidence for structural changes in the a subunit accompanying E1-E2 transition in Na,K-ATPase [52]. Localization of the proteolytic splits provided a shortcut to identification of residues involved in E1-E2 transition [33,53,54] and to detection of structure-function correlations [33]. Further proteolysis identifies segments at the surface of the protein and as the cytoplasmic protrusion is shaved off all ATP-dependent reactions are abolished. 

Transition from the high-energy phosphoform E]P[3Na] to the K-sensitive E2P[2Na] of Na,K-ATPase are accompanied by conformational transitions in protein structure and changes of the capacity and orientation of cation sites. In the Ej form of Na,K-ATPase, the exposure of Chys (Leu ) and Trys (Arg ) to cleavage reflects that the cation sites of the phosphoprotein are in a conformation oriented towards the cytoplasm with a capacity for occlusion of three Na ions. The E2 form... 

Thus, transition from Ei to E2 consists of an integrated structural change involving protection of bond C3 or T3 in the second cytoplasmic domain and exposure of T in the central domain, while the position of T2 in the N-terminus is altered relative to the central domain (Tj) so that cleavage of T2 becomes secondary to cleavage of T1 within the same a subunit in the E2 form. 

C3 cleavage is a selective and particularly efficient tool for examining structure-function relationships of the second cytoplasmic domain. Binding affinities for ADP and ATP are reduced 4-5-fold, while TNP-ATP binds with the same affinity as in native Na,K-ATPase. Nucleotide binding is not affected by or Rb although... 

Fig. 2. The hypothetical structure of Ca -ATPase. The structure consists of three major cytoplasmic domains, a pentahelical stalk region, and an intramembranous domain with ten, presumably helical, transmembrane segments. Ti and T2 mark the tryptic cleavage sites. Inset charged amino acids in and near the transmembrane region that may contribute to a Ca " -channel. Adapted from Brandi et al. [8].

Production of Malonyl-CoA for the Fatty Acid Biosynthesis. Acetyl-CoA serves as a substrate in the production of malonyl-CoA. There are several routes by which acetyl-CoA is supplied to die cytoplasm. One route is the transfer of acetyl residues from the mitochondrial matrix across the mitochondrial membrane into the cyto-plasm. This process resembles a fatty acid transport and is likewise effected with the participation of carnitine and the enzyme acetyl-CoA-camitine transferase. Another route is the production of acetyl-CoA from citrate. Citrate is delivered from the mitochondria and undergoes cleavage in the cytoplasm by the action of the enzyme ATP-citrate lyase ... 

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