Yves Benyamin

Alpha actinin–CapZ, an anchoring complex for thin filaments in Z-line

CapZ is a widely distributed and highly conserved, heterodimeric protein, that nucleates actin polymerization and binds to the barbed ends of actin filaments, preventing the addition or loss of actin monomers. CapZ interaction with actin filaments was shown to be of high affinity and decreased in the presence of PIP2. CapZ was located in nascent Z-lines during skeletal muscle myofibrillogenesis before the striated appearance of thin filaments in sarcomers. In this study, the stabilization and the anchorage of thin filaments were explored through identification of CapZ partners in the Z-line. Fish (sea bass) striated white muscle and its related Z-line proteins were selected since they correspond to the simplest Z-line organization. We report here the interaction between purified CapZ and α-actinin, a major component of Z filaments and polar links in Z-discs. Affinity of CapZ for α-actinin, estimated by fluorescence and immunochemical assays, is in the μ m range. This association was found to be independent of actin and shown to be weakened in the presence of phosphoinositides. Binding contacts on the α-actinin molecule lie in the 55 kDa repetitive domain. A model including CapZ/α-actinin/titin/actin interactions is proposed considering Luthers 3D Z-line reconstruction.

Calpain involvement in the remodeling of cytoskeletal anchorage complexes.

Cells offer different types of cytoskeletal anchorages: transitory structures such as focal contacts and perennial ones such as the sarcomeric cytoskeleton of muscle cells. The turnover of these structures is controlled with different timing by a family of cysteine proteases activated by calcium, the calpains. The large number of potential substrates present in each of these structures imposes fine tuning of the activity of the proteases to avoid excessive action. This phenomenon is thus guaranteed by various types of regulation, ranging from a relatively high calcium concentration necessary for activation, phosphorylation of substrates or the proteases themselves with either a favorable or inhibitory effect, possible intervention of phospholipids, and the presence of a specific inhibitor and its possible degradation before activation. Finally, formation of multiprotein complexes containing calpains offers a new method of regulation.

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium‐dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097–2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z‐band and at the midpoint of the I‐band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I‐band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N‐terminal Z8–I5 region and the N1‐line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl2 and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z‐band edge and the N1‐line as well as at the N2‐line level, two sarcomeric regions where early postmortem proteolysis is detected.

Identification of digestive proteinases of Penaeus kerathurus (Forskål): A comparison with Penaeus japonicus Bate

Abstract 1. 1. The proteolytic activity of digestive gland of Penaeus kerathurus was investigated using natural substrates. Effects of temperature, pH, metallic. ions, chelators and iodoacetamide were studied. 2. 2. Trypsin, carboxypeptidases A and B and leucine aminopeptidase activities were identified in shrimps P. kerathurus and P. japonicus . A very poor chymotrypsin-like activity was found only in P. kerathurus . No elastolytic activity but a collagenase-like one was detected. 3. 3. The approximate mol. wts of trypsin and carboxypeptidases A and B were respectively 25,000 and 35,000. A substantial proteolytic activity was observed at a low molecular weight around 11,000 daltons. 4. 4. The isozymic distribution of endo- and exopeptidases secreted by the hepatopancreas of the two species was analysed by electrophoretic pattern.

Post mortem release of fish white muscle α-actinin as a marker of disorganisation

alpha-Actinin release and its degradation from myofibrils Z-line were studied in post mortem white dorsal muscle from bass and sea trout stored at 4 degrees C and 10 degrees C. Using alpha-actinin specific antibodies, we show that this protein is rapidly released within the first 24 h for the two species, and reaches a plateau within 4 days. Proteolysis take place very rapidly in bass muscle yielding 80 and 40 kDa fragments from alpha-actinin as major bands of proteolysis. Sea trout muscle is more resistant, and muscle stored at 4 degrees C is not significantly alpha-actinin degraded even 10 days after death. In the case of sea trout muscle stored at 10 degrees C, an increasing quantity of 80 and 40 kDa fragment can be observed after the third day. These results show that release and proteolysis of alpha-actinin are time- and temperature-dependent processes that take place at the early stages of fish storage. Furthermore, we observed that proteolysis of alpha-actinin seems to be dependent on fish species. In both species studied, the early release of alpha-actinin comes before the degradation of released molecules, and appears as a biphasic process throughout the disorganisation of post mortem muscle in fish cold-stored above 0 degree C.

Localization of a new α-actinin binding site in the COOH — terminal part of actin sequence

Summary The interaction of filamentous actin with α-actinin, an actin cross-linking protein, is well established. On the other hand, monomeric actin — α-actinin interaction has been a subject of controversy. In this report, we have characterized the interaction of monomeric actin, coated on plastic plates under conditions of non — polymerizatioon, with α-actinin in presence of magnesium. Using specific polyclonal anti — actin antibodies, with the whole molecule or purified peptides, we have localized two sites of interaction on actin module: one near Thr-103 and a new one in the twenty last amino acids.

Cation binding sites on actin: A structural relationship between antigenic epitopes and cation exchange

Divalent cations such as Mg2+ and Ca2+, which bind specifically to actin, induce conformational changes that affect its antigenic structure. The distribution of antigenic epitopes on the sequence shows that these structural modifications involve epitopes related to monomer-monomer interfaces. In the N-terminal part, the 1-7 acidic extremity is not affected, in contrast with sequence 18-28. The ability of polycations such as diamine to modify the actin structure at concentrations below 0.1 microM strengthens the hypothesis that in vivo these compounds act locally and specifically on actin polymerization.

Fish muscle cytoskeleton integrity is not dependent on intact thin filaments

Striated musclecytoskeleton was studied by ultrastructure and electrophoresis.Treatment of sea bass white muscle myofibrils and glycerinatedfibres with calpain caused disruption of costameres, intermediatefilaments, and Z-line, without altering sarcomeres. V8 proteasealso caused loss of costameres and Z-line, and disruptedsarcomeres without affecting the intermediate filaments.Recombinant lipase caused loss of Z-lines and also sarcolemmadetachment, without changing sarcomeres or intermediatefilaments. DNase-1 removed thin filaments and partially removedZ-lines while leaving intact the sarcolemma attachments andintermediate filaments. Calpain, V8 protease, lipase and DNase-1treatments induced extensive loss of α-actinin from the Z-line, which could be related to titin cleavage (calpain, V8),phosphoinositide hydrolysis (lipase), and actin depolymerisation(DNase-1). These results show that the cytoskeletal componentsare independent of intact thin filaments

The identification of a second cofilin binding site on actin suggests a novel, intercalated arrangement of F-actin binding.

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112–125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37–44). We propose that cofilin binds “behind” one monomer and “in front” of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771–781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047–1055).

Sarcomeric disorganization in post-mortem fish muscles.

1. The post-mortem evolution of protein pattern in fish striated muscle was followed by SDS-PAGE, after different conditions of storage time and temperature. 2. Sarcoplasmic and sarcomeric fractions were analyzed respectively by low and high ionic strength extractions of fish muscle samples. 3. No evident modification of electrophoretic patterns was observed during the pre-rigor mortis period. 4. The high mol. wt proteins titin and nebulin were highly sensitive to proteolysis during the rigor mortis period. 5. Myosin extraction was predominantly influenced by the storage temperature. The myosin content of the extracts decreased during the rigor mortis period at storage temperatures greater than 8 degrees C. 6. alpha-Actinin was very resistant to proteolysis, but could be released from Z-disc structure during post-mortem aging.