scholarly journals Bioinspired Design of Lysolytic Triterpenoid-peptide Conjugates that Kill African Trypanosomes

2018 ◽  
Author(s):  
W.-Matthias Leeder ◽  
Fabian Giehler ◽  
Juliane Joswig ◽  
H. Ulrich Göringer
Keyword(s):  
Protein Complexes ◽  
Building Blocks ◽  
Optimized Design ◽  
Natural Immunity ◽  
Cell Binding ◽  
Peptide Conjugates ◽  
African Trypanosomes ◽  
Stepwise Development ◽  
A Cell ◽  
The Many

AbstractHumans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). Active TLF-ingredient is the primate-specific apolipoprotein L1 (ApoL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies only two are able to counteract the activity of ApoL1, which illustrates its evolutionary optimized design and trypanocidal potency. Here we ask the question whether a synthetic (syn)TLF can be synthesized using the design principles of the natural TLF-complexes but relying on different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell binding, uptake and lysosomal transport-moduls and the synthetic peptide GALA as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis demonstrating proof-of-principle for the bioinspired, forward-design of a synTLF.

Structural features and functional domains of amassin-1, a cell-binding olfactomedin protein

2007 ◽  
Vol 85 (5) ◽  
pp. 552-562 ◽  
Author(s):  
Brian J. Hillier ◽  
Victor D. Vacquier
Keyword(s):  
Disulfide Bonds ◽  
Biological Activities ◽  
Structural Features ◽  
Body Cavity ◽  
Binding Activity ◽  
Coiled Coils ◽  
Cell Binding ◽  
Calcium Dependent ◽  
Dependent Cell ◽  
A Cell

Amassin-1 mediates a rapid cell adhesion that tightly adheres sea urchin coelomocytes (body cavity immunocytes) together. Three major structural regions exist in amassin-1: a short β region, 3 coiled coils, and an olfactomedin domain. Amassin-1 contains 8 disulfide-bonded cysteines that, upon reduction, render it inactive. Truncated forms of recombinant amassin-1 were expressed and purified from Pichia pastoris and their disulfide bonding and biological activities investigated. Expressed alone, the olfactomedin domain contained 2 intramolecular disulfide bonds, existed in a monomeric state, and inhibited amassin-1-mediated clotting of coelomocytes by a calcium-dependent cell-binding activity. The N-terminal β region, containing 3 cysteines, was not required for clotting activity. The coiled coils may dimerize amassin-1 in a parallel orientation through a homodimerizing disulfide bond. Neither amassin-1 fragments that were disulfide-linked as dimers or that were engineered to exist as dimers induced coelomocytes clotting. Clotting required higher multimeric states of amassin-1, possibly tetramers, which occurred through the N-terminal β region and (or) the first segment of coiled coils.

scholarly journals The Protective Role of Dormant Origins in Response to Replicative Stress

2018 ◽  
Vol 19 (11) ◽  
pp. 3569 ◽  
Author(s):  
Lilas Courtot ◽  
Jean-Sébastien Hoffmann ◽  
Valérie Bergoglio
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Replication Timing ◽  
Protective Role ◽  
Entire Genome ◽  
Replicative Stress ◽  
A Cell ◽  
The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

scholarly journals Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum.

1988 ◽  
Vol 107 (5) ◽  
pp. 1835-1843 ◽  
Author(s):  
R K Kamboj ◽  
L M Wong ◽  
T Y Lam ◽  
C H Siu
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Fusion Proteins ◽  
Binding Activity ◽  
Binding Domain ◽  
Globular Domain ◽  
Cell Binding ◽  
Homophilic Interaction ◽  
A Cell ◽  
Cell Binding Domain

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.

In Vitro Removal of Ochratoxin A by Wine Lactic Acid Bacteria

2007 ◽  
Vol 70 (9) ◽  
pp. 2155-2160 ◽  
Author(s):  
VINCENZO DEL PRETE ◽  
HECTOR RODRIGUEZ ◽  
ALFONSO V. CARRASCOSA ◽  
BLANCA de las RIVAS ◽  
EMILIA GARCIA-MORUNO ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Ochratoxin A ◽  
High Performance ◽  
Culture Medium ◽  
Degradation Products ◽  
Cell Binding ◽  
A Cell ◽  
In Vitro Interaction

A study was carried out to determine the in vitro interaction between ochratoxin A (OTA) and wine lactic acid bacteria (LAB). Fifteen strains belonging to five relevant oenological LAB species were grown in liquid synthetic culture medium containing OTA. The portion of OTA removed during the bacterial growth was 8 to 28%. The OTA removed from the supernatants was partially recovered (31 to 57%) from the bacterial pellet. Cell-free extracts of three representative strains were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade OTA was studied. OTA was not degraded by cell-free extracts of wine LAB strains, and no degradation products of OTA were detected in the high-performance liquid chromatograms of the methanol extract of the bacterial pellet. On the basis of these results, we conclude that OTA removal by wine LAB is a cell-binding phenomenon. The chemistry and the molecular basis of OTA binding to wine LAB remains unknown.

scholarly journals The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

2014 ◽  
Vol 14 (3) ◽  
pp. 206-215 ◽  
Author(s):  
Steven Sykes ◽  
Anthony Szempruch ◽  
Stephen Hajduk
Keyword(s):  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Krebs Cycle ◽  
Content Type ◽  
African Trypanosomes ◽  
Atp Production ◽  
Cycle Enzyme ◽  
A Cell ◽  
Direct Localization ◽  
Green Fluorescent

ABSTRACT α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei . The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei . These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei .

scholarly journals A Cell-free Assay for Glycosylphosphatidylinositol Anchoring in African Trypanosomes

1999 ◽  
Vol 274 (23) ◽  
pp. 16479-16486 ◽  
Author(s):  
Deepak K. Sharma ◽  
Jolanta Vidugiriene ◽  
James D. Bangs ◽  
Anant K. Menon
Keyword(s):  
African Trypanosomes ◽  
A Cell

In vitro incorporation of a cell-binding protein to a lentiviral vector using an engineered split intein enables targeted delivery of genetic cargo

2015 ◽  
Vol 112 (12) ◽  
pp. 2611-2617 ◽  
Author(s):  
Ana M. Chamoun-Emanuelli ◽  
Gus Wright ◽  
Smith Roger ◽  
Robert C. Münch ◽  
Christian J. Buchholz ◽  
...  
Keyword(s):  
Targeted Delivery ◽  
Lentiviral Vector ◽  
Binding Protein ◽  
Cell Binding ◽  
Split Intein ◽  
A Cell

scholarly journals Optimal transcriptional regulation of cellular responses to sudden environmental shifts

2020 ◽  
Author(s):  
Daniel Schultz ◽  
Lev S. Tsimring
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Cell Response ◽  
Cellular Responses ◽  
Natural Systems ◽  
Selective Pressures ◽  
Regulatory Strategies ◽  
Regulatory Strategy ◽  
A Cell ◽  
The Many

ABSTRACTCellular responses to sudden changes in their environment require prompt expression of the correct levels of the appropriate enzymes. These enzymes are typically regulated by transcription factors that sense the presence of inducers and control gene expression for the duration of the response. The specific choice of regulatory strategy depends on the characteristics of each cell response, with the pattern of gene expression dictated by parameters such as the affinity of the transcription factor to its binding sites and the strength of the promoters it regulates. Although much is known about how gene regulation determines the dynamics of cell responses, we still lack a framework to understand how the many different regulatory strategies evolved in natural systems relate to the constraints imposed by the selective pressures acting in each particular case. Here, we analyze a dynamical model of a cell response where expression of a transcriptionally repressed enzyme is induced by a sudden exposure to its substrate. We identify strategies of gene regulation that optimize the response for different types of selective pressures, which we define as a set of costs associated with substrate, enzyme and repressor intracellular concentrations during the response. We find that regulated responses happen within a defined region in the parameter space. While responses to costly (toxic) substrates favor the usage of strongly self-regulated repressors, responses where expression of enzyme is more costly than its substrate favor the usage of constitutively expressed repressors. There is only a very narrow range of selective pressures that would favor weakly self-regulated repressors. This framework can be used to infer which costs and benefits are most critical in the evolution of natural examples of cellular responses, and to predict how a response can optimize its regulation when transported to a new environment with different demands.

scholarly journals Myosins generate contractile force and maintain organization in the cytokinetic contractile ring

2021 ◽  
Author(s):  
Zachary A. McDargh ◽  
Shuyuan Wang ◽  
Harvey F. Chin ◽  
Sathish Thiyagarajan ◽  
Erdem Karatekin ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Striated Muscle ◽  
Protein Complexes ◽  
Myosin Ii ◽  
Force Production ◽  
Super Resolution ◽  
Building Blocks ◽  
Contractile Ring ◽  
Ring Model ◽  
Self Assembling

During cytokinesis, cells assemble an actomyosin contractile ring whose tension constricts and divides cells, but the ring tension was rarely measured. Actomyosin force generation is well understood for the regular sarcomeric architecture of striated muscle, but recent super-resolution studies of fission yeast contractile rings revealed organizational building blocks that are not sarcomeres but irregularly positioned plasma membrane-anchored protein complexes called nodes. Here, we measured contractile ring tensions in fission yeast protoplast cells. The myosin II isoforms Myo2 and Myp2 generated the tension, with a ~2-fold greater contribution from Myo2. Simulations of a molecularly detailed ring model revealed a sliding node mechanism for tension, where nodes hosting tense actin filaments were pulled bidirectionally around the ring. Myo2 and Myp2 chaperoned self-assembling components into the ring organization, and anchored the ring against bridging instabilities. Thus, beyond force production, Myo2 and Myp2 are the principal organizers, bundlers and anchors of the contractile ring.

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