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1.
Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ∼190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.  相似文献   

2.
The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.  相似文献   

3.
Tropomyosin (Tpm) is an extended α-helical coiled-coil homodimer that regulates actinomyosin interactions in muscle. Molecular simulations of four Tpms, two from the vertebrate class Mammalia (rat and pig), and two from the invertebrate class Malacostraca (shrimp and lobster), showed that despite extensive sequence and structural homology across metazoans, dynamic behavior—particularly long-range structural fluctuations—were clearly distinct. Vertebrate Tpms were more flexible and sampled complex, multi-state conformational landscapes. Invertebrate Tpms were more rigid, sampling a highly constrained harmonic landscape. Filtering of trajectories by principle component analysis into essential subspaces showed significant overlap within but not between phyla. In vertebrate Tpms, hinge-regions decoupled long-range interhelical motions and suggested distinct domains. In contrast, crustacean Tpms did not exhibit long-range dynamic correlations—behaving more like a single rigid rod on the nanosecond time scale. These observations suggest there may be divergent mechanisms for Tpm binding to actin filaments, where conformational flexibility in mammalian Tpm allows a preorganized shape complementary to the filament surface, and where rigidity in the crustacean Tpm requires concerted bending and binding.  相似文献   

4.
Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.  相似文献   

5.
The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.  相似文献   

6.
The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three isoforms when expressed in the Escherichia coli strain BL-21(DE3). These isoforms are produced by the non-homogeneous processing of the N-terminal methionine residue by the endogenous bacterial aminopeptidase and differ in the first residue (1-met, 1-ala, and 1-pro). Importantly, these isoforms have significantly different binding affinities for the IL receptor protein. Varying the temperature, media composition, and point of induction affects this N-terminal processing to favor one of the three isoforms of IL-1beta. We found changes in media composition and/or point of induction affected the abundance of the isoforms by as much as 15-fold. The 1-pro isoform decreased from 60.9 to 4.7% in Luria broth (LB) and minimal media (MM), respectively, when protein expression was induced at an OD600 of 0.9. Conversely, the abundance of the 1-met isoform is much higher in MM than in LB showing the reverse effect (2.6 and 50.7% in LB and MM, respectively, at an OD600 of 0.9), and the degree to which they are favored depends significantly upon the induction point. Our results show that it is possible to favor the expression of various N-terminal isoforms of IL-1beta by adjusting the environmental growth conditions. Given that the initiator methionine residue is necessary for expression in bacterial hosts and is known to affect the stability of other recombinant proteins our approach may be a useful general method for determining the optimal conditions for expressing and purifying pure, homogenous samples of recombinant proteins for structural and biological studies.  相似文献   

7.
Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.  相似文献   

8.
Injury to lens epithelial cells (LECs) leads to epithelial–mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α‐smooth muscle actin (α‐SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up‐regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non‐cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α‐SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm‐based inhibitors for postponing PCO and cataractogenesis.  相似文献   

9.
Lamina associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is ubiquitously expressed. LAP1 binds to lamins and chromatin, probably contributing to the maintenance of the nuclear envelope architecture. Moreover, LAP1 also interacts with torsinA and emerin, proteins involved in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy disorder, respectively. Given its relevance to human pathological conditions, it is important to better understand the functional diversity of LAP1 proteins. In rat, the LAP1 gene (TOR1AIP1) undergoes alternative splicing to originate three LAP1 isoforms (LAP1A, B and C). However, it remains unclear if the same occurs with the human TOR1AIP1 gene, since only the LAP1B isoform had thus far been identified in human cells. In silico analysis suggested that, across different species, potential new LAP1 isoforms could be generated by alternative splicing. Using shRNA to induce LAP1 knockdown and HPLC-mass spectrometry analysis the presence of two isoforms in human cells was described and validated: LAP1B and LAP1C; the latter is putatively N-terminal truncated. LAP1B and LAP1C expression profiles appear to be dependent on the specific tissues analyzed and in cultured cells LAP1C was the major isoform detected. Moreover, LAP1B and LAP1C expression increased during neuronal maturation, suggesting that LAP1 is relevant in this process. Both isoforms were found to be post-translationally modified by phosphorylation and methionine oxidation and two LAP1B/LAP1C residues were shown to be dephosphorylated by PP1. This study permitted the identification of the novel human LAP1C isoform and partially unraveled the molecular basis of LAP1 regulation.  相似文献   

10.
Almost all proteins are expressed in several variants, also known as isoforms. Individual protein variants differ by modifications of the individual amino acid side chains, or the N- or C-terminus. Typical modifications are glycosylation, phosphorylation, acetylation, methylation, deamidation or oxidation. It is of utmost interest to either get a quantitative picture of the variants of a particular protein or to separate the variants in order to be able to identify their molecular structure. Protein variants are present in native as well as in recombinant proteins. In the case of protein production it is interesting, how variants are generated during fermentation, purification processes, storage, and how present individual variants influence the biological activity. This review provides a comparison of chromatographic and electrophoretic separation methods to analyze and to prepare protein variants.  相似文献   

11.
We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.  相似文献   

12.
Zhang J  Zhang H  Ayaz-Guner S  Chen YC  Dong X  Xu Q  Ge Y 《Biochemistry》2011,50(27):6081-6092
Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca(2+)-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.  相似文献   

13.
Myosin II, the conventional two-headed myosin that forms bipolar filaments, is directly involved in regulating cytokinesis, cell motility and cell morphology in nonmuscle cells. To understand the mechanisms by which nonmuscle myosin-II regulates these processes, investigators are now looking at the regulation of this molecule in vertebrate nonmuscle cells. The identification of multiple isoforms of nonmuscle myosin-II, whose activities and regulation differ from that of smooth muscle myosin-II, suggests that, in addition to regulatory light chain phosphorylation, other regulatory mechanisms control vertebrate nonmuscle myosin-II activity.  相似文献   

14.
The majority of eukaryotic proteins are subjected to N-terminal acetylation (Nt-acetylation), catalysed by N-terminal acetyltransferases (NATs). Recently, the structure of an NAT-peptide complex was determined, and detailed proteome-wide Nt-acetylation patterns were revealed. Furthermore, Nt-acetylation just emerged as a multifunctional regulator, acting as a protein degradation signal, an inhibitor of endoplasmic reticulum (ER) translocation, and a mediator of protein complex formation. Nt-acetylation is regulated by acetyl-coenzyme A (Ac-CoA) levels, and thereby links metabolic cell states to cell death. The essentiality of NATs in humans is stressed by the recent discovery of a human hereditary lethal disease caused by a mutation in an NAT gene. Here, we discuss how these recent findings shed light on NATs as major protein regulators and key cellular players.  相似文献   

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19.
We have investigated accumulation of alpha-actinin, the principal cross-linker of actin filaments, in four Drosophila fliA mutants. A single gene is variably spliced to generate one nonmuscle and two muscle isoforms whose primary sequence differences are confined to a peptide spanning the actin binding domain and first central repeat. In fliA3 the synthesis of an adult muscle-specific isoform is blocked in flight and leg muscles, while in fliA4 the synthesis of nonmuscle and both muscle-specific isoforms is severely reduced. Affected muscles are weak or paralyzed, and, in the case of fliA3, atrophic. Their myofibrils, while structurally irregular, are remarkably normal considering that they are nearly devoid of a major contractile protein. Also surprising is that no obvious nonmuscle cell abnormalities can be discerned despite the fact that both the fliA1- and fliA4-associated mutations perturb the nonmuscle isoform. Our observations suggest that alpha-actinin stabilizes and anchors thin filament arrays, rather than orchestrating their assembly, and further imply that alpha-actinin function is redundant in both muscle and nonmuscle cells.  相似文献   

20.
A single human myosin light chain kinase gene (MLCK; MYLK)   总被引:7,自引:0,他引:7  
Lazar V  Garcia JG 《Genomics》1999,57(2):256-267
The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garcia et al., 1997, Am. J. Respir. Cell Mol. Biol. 16, 489-494) except for a deletion of nucleotides 1428-1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081-5233 (MLCK 3a, D3), double deletions of nucleotides 1428-1634 and 5081-5233 (MLCK 3b), and nucleotide deletions 4534-4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5' and 3' neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.  相似文献   

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