scholarly journals Dual binding motifs underpin the hierarchical association of perilipins1–3 with lipid droplets

2019 ◽  
Vol 30 (5) ◽  
pp. 703-716 ◽  
Author(s):  
Dalila Ajjaji ◽  
Kalthoum Ben M'barek ◽  
Michael L. Mimmack ◽  
Cheryl England ◽  
Haya Herscovitz ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Tissue Type ◽  
Binding Motifs ◽  
Amino Terminal ◽  
A Cell ◽  
Oil Water ◽  
Carboxy Terminal ◽  
Hierarchical Manner

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a “hierarchical” manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1–3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both “naked” and phospholipid-coated oil–water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1–3 to LDs.

scholarly journals Identification and Characterization of a Novel Serine-Arginine-Rich Splicing Regulatory Protein

2000 ◽  
Vol 20 (9) ◽  
pp. 3049-3057 ◽  
Author(s):  
Daron C. Barnard ◽  
James G. Patton
Keyword(s):  
Regulatory Protein ◽  
Structural Similarity ◽  
Sr Proteins ◽  
Rna Recognition Motif ◽  
Sr Protein ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Rs Domains

ABSTRACT We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

scholarly journals Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation.

1987 ◽  
Vol 7 (12) ◽  
pp. 4280-4289 ◽  
Author(s):  
A M Pendergast ◽  
J A Traugh ◽  
O N Witte
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Amino Terminal ◽  
Kinase C ◽  
Carboxy Terminal

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

scholarly journals The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

1998 ◽  
Vol 18 (12) ◽  
pp. 7466-7477 ◽  
Author(s):  
Soundarapandian Velmurugan ◽  
Yong-Tae Ahn ◽  
Xian-Mei Yang ◽  
Xu-Li Wu ◽  
Makkuni Jayaram
Keyword(s):  
Plasmid Stability ◽  
High Specificity ◽  
Nuclear Localization Sequence ◽  
Nuclear Targeting ◽  
Amino Terminal ◽  
Rep Proteins ◽  
Localization Sequence ◽  
Carboxy Terminal

ABSTRACT The stable inheritance of the 2μm plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with thecis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2μm circle-derived plasmid shows relatively poor stability.

Structure, function and biology of tissue factor pathway inhibitor-2

2005 ◽  
Vol 94 (12) ◽  
pp. 1122-1130 ◽  
Author(s):  
Hitendra S. Chand ◽  
Donald C. Foster ◽  
Walter Kisiel
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Serine Proteinase ◽  
In Vivo Studies ◽  
Amino Terminal ◽  
Tissue Factor Pathway ◽  
Kunitz Type ◽  
Carboxy Terminal

SummaryTissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region, three tandem Kunitz-type domains and a positively charged carboxy-terminal tail. Human TFPI-2, previously designated as placental protein 5, inhibits a broad spectrum of serine proteinases almost exclusively through its first Kunitz-type domain, and is thought to play an important role in the regulation of extracellular matrix digestion and re-modeling. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth/metastasis. In this review, we document current information regarding the expression of TFPI-2 by various tissues, its inhibitory activity and proteinase specificity in-vitro, and discuss possible physiological roles for this inhibitor based on in-vivo studies.

scholarly journals Effect of deletion mutation on the recombination activity of Cre recombinase.

2005 ◽  
Vol 52 (2) ◽  
pp. 541-544 ◽  
Author(s):  
Liu Rongrong ◽  
Wang Lixia ◽  
Lin Zhongping
Keyword(s):  
Cre Recombinase ◽  
Terminal Deletion ◽  
Middle Region ◽  
Wild Type ◽  
Recombination Activity ◽  
Bacteriophage P1 ◽  
Amino Terminal ◽  
Carboxy Terminal

Cre recombinase from bacteriophage P1 is widely used in both in vitro and in vivo DNA manipulations. Based on a structural and functional analysis, three deleted cre mutants were constructed and expressed in Escherichia coli. Mutated recombinases were purified and their recombination activities were determined in vitro. Our results revealed that the mutant with amino-terminal deletion retains the recombination activity as high as wild type Cre; however, the carboxy-terminal deletion and the middle region deletion both lead to a complete loss of the recombinase function.

Assembly of A- and B-type lamins studied in vivo with the baculovirus system

1997 ◽  
Vol 110 (20) ◽  
pp. 2519-2532 ◽  
Author(s):  
M. Klapper ◽  
K. Exner ◽  
A. Kempf ◽  
C. Gehrig ◽  
N. Stuurman ◽  
...  
Keyword(s):  
Sf9 Cells ◽  
Lamin A ◽  
Nuclear Compartment ◽  
Amino Terminal ◽  
Filament Bundles ◽  
Baculovirus System ◽  
Carboxy Terminal ◽  
Triton X

We have expressed an A-type lamin (Xenopus lamin A), a probable A-type lamin (Drosophila lamin C), two B-type lamins (Xenopus lamin LI, Drosophila lamin Dmo), and two mutants of Xenopus lamin A in Sf9 cells. All proteins were synthesized at high levels resulting in formation of paracrystals with an axial repeat of 18.5-20.0 nm by A-type lamins; in contrast B-type lamins assembled into aggregates with a fibrillar ultrastructure. Of the four wild-type proteins analyzed only lamin Dmo was found in the nuclear compartment of Sf9 cells in association with the lamina whereas the three other lamins assembled into polymers localized in the cytoplasm as well as the nucleoplasm. The Xenopus lamin A mutant lacking the complete carboxy-terminal tail assembled in the cytoplasm into long filament bundles consisting of fibrils of less than 6 nm diameter. In vitro the non-helical amino-terminal head domain of lamins is required for the formation of ‘head-to-tail’ polymers. A lamin A mutant lacking this domain could be efficiently extracted from Sf9 cells with physiological buffers containing Triton X-100, demonstrating the importance of this domain for lamin assembly in vivo.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation

1987 ◽  
Vol 7 (12) ◽  
pp. 4280-4289
Author(s):  
A M Pendergast ◽  
J A Traugh ◽  
O N Witte
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Amino Terminal ◽  
Kinase C ◽  
Carboxy Terminal

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

scholarly journals Mutations That Alter the Kinase and Phosphatase Activities of the Two-Component Sensor EnvZ

1998 ◽  
Vol 180 (17) ◽  
pp. 4538-4546 ◽  
Author(s):  
Weihong Hsing ◽  
Frank D. Russo ◽  
Karen K. Bernd ◽  
Thomas J. Silhavy
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Terminal ◽  
Medium Osmolarity ◽  
Two Component ◽  
Sensor Kinases ◽  
Dominant State ◽  
Carboxy Terminal

ABSTRACT EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in Escherichia coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K+ P−) and the OmpR-P phosphatase-dominant state (K− P+). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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