scholarly journals The unique tropism ofMycobacterium lepraeto the nasal epithelial cells can be explained by the mammalian cell entry protein 1A

2018 ◽  
Author(s):  
Viesta Beby Fadlitha ◽  
Fuki Yamamoto ◽  
Irfan Idris ◽  
Haslindah Dahlan ◽  
Naoya Sato ◽  
...  
Keyword(s):  
Amino Acids ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Recombinant Proteins ◽  
Cell Entry ◽  
Important Region ◽  
C Terminus ◽  
Protein Encoding ◽  
N Terminus ◽  
Encoding Genes

AbstractLeprosy is a chronic infection where the skin and peripheral nervous system is invaded byMycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet forM.leprae.Mce1A protein (442 aa) is coded by mce1A (1326 bp) ofM.leprae. The mce1A homolog inMycobacterium tuberculosisis known to be associated withM.tuberculosisepithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that mce1A ofM.lepraeis also associated with epithelial cell entry. This study is aimed at identifying particular sequences within mce1A associated withM.lepraeepithelial cell entry.Recombinant proteins having N-terminus and C-terminus truncations of the mce1A region ofM.lepraewere created inEschericia coli.Entry activity of latex beads, coated with these truncated proteins (r-lep37kDa and r-lep27kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316 – 921 bp region was divided into three sub-regions: 316 – 531 bp (InvX), 532 – 753 bp (InvY), and 754 – 921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface ofE.coli.Entry of theseE.coliinto monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. OnlyE.coliharboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa - InvXd, containing sequences 1 – 24 aa, 25 – 46 aa, 47 – 57 aa, and 58 – 72 aa, respectively.RecombinantE.coli, expressing each of InvXa - InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of mce1A are important forM.lepraeinvasion into nasal epithelial cells.Author SummaryMce1A protein is encoded by the mce1A region of mce1 locus ofM.tuberculosisandM.leprae, and is involved in the bacteria’s invasion into epithelial cells. The present study revealed that the active sequence ofM.lepraeinvolved in the invasion into nasal mucosa epithelial cells is present in the 316-531 bp region of mce1A.The most important region of mce1A protein involved in the invasion ofM.tuberculosisinto human epithelial cells is called the InvIII region, which is located between amino acids at position 130 to 152. The InvIII region ofM.tuberculosiscorresponds to InvXb ofM.leprae. The sequences of these regions are identical between amino acids at positions 10 to position 22 as counted from the N terminus, except that amino acids at positions 1 to 3, 5, 8, 9, 13 are different betweenM.lepraeandM.tuberculosis. Suppression test results also indicated that the most important region of mce1A protein ofM.lepraeinvolved in the invasion into human epithelial cells is different from thatM.tuberculosis. WhileM.tuberculosishas 3,959 protein-encoding genes and only 6 pseudogenes,M.lepraehas only 1,604 protein-encoding genes but has 1,116 pseudogenes indicating that inM.leprae, far more proteins are inactivated as compared toM.tuberculosis. The present study also revealed that, as inM.tuberculosis,the mce1A protein is expressed on the surface of bacteria as a native protein. In light of these data, the mce1A protein is considered to be one of the most important proteins involved in the invasion ofM.lepraeinto nasal mucosa epithelial cells.

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

scholarly journals Transcriptome Analysis Unveils the Effects of Proline on Gene Expression in the Yeast Komagataella phaffii

2021 ◽  
Vol 10 (1) ◽  
pp. 67
Author(s):  
Andrey Rumyantsev ◽  
Anton Sidorin ◽  
Artemii Volkov ◽  
Ousama Al Shanaa ◽  
Elena Sambuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Recombinant Proteins ◽  
Gene Promoter ◽  
Energy Sources ◽  
Experimental Conditions ◽  
Komagataella Phaffii ◽  
Protein Encoding ◽  
Modern Biotechnology ◽  
Encoding Genes ◽  
Transcriptome Level

Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

Plakoglobin domains that define its association with the desmosomal cadherins and the classical cadherins: identification of unique and shared domains

1996 ◽  
Vol 109 (5) ◽  
pp. 1143-1154 ◽  
Author(s):  
J.K. Wahl ◽  
P.A. Sacco ◽  
T.M. McGranahan-Sadler ◽  
L.M. Sauppe ◽  
M.J. Wheelock ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intermediate Filament ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Central Domain ◽  
Desmosomal Cadherins ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Classical Cadherins ◽  
N Terminus

Two cell-cell junctions, the adherens junction and the desmosome, are prominent in epithelial cells. These junctions are composed of transmembrane cadherins which interact with cytoplasmic proteins that serve to link the cadherin to the cytoskeleton. One component of both adherens junctions and desmosomes is plakoglobin. In the adherens junction plakoglobin interacts with both the classical cadherin and with alpha-catenin. Alpha-catenin in turn interacts with microfilaments. The role plakoglobin plays in the desmosome is not well understood. Plakoglobin interacts with the desmosomal cadherins, but how and if this mediates interactions with the intermediate filament cytoskeleton is not known. Here we compare the domains of plakoglobin that allow it to associate with the desmosomal cadherins with those involved in interactions with the classical cadherins. We show that three sites on plakoglobin are involved in associations with the desmosomal cadherins. A domain near the N terminus is unique to the desmosomal cadherins and overlaps with the site that interacts with alpha-catenin, suggesting that there may be competition between alpha-catenin and the desmosomal cadherins for interactions with plakoglobin. In addition, a central domain is shared with regions used by plakoglobin to associate with the classical cadherins. Finally, a domain near the C terminus is shown to strongly modulate the interactions with the desmosomal cadherins. This latter domain also contributes to the association of plakoglobin with the classical cadherins.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Ian C. Fields ◽  
Shelby M. King ◽  
Elina Shteyn ◽  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Amino Acid Residues ◽  
Recycling Endosomes ◽  
C Terminus ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

scholarly journals The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16

2017 ◽  
Vol 28 (18) ◽  
pp. 2420-2433 ◽  
Author(s):  
Yuqing Hou ◽  
George B. Witman
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Intraflagellar Transport ◽  
Critical Importance ◽  
C Terminus ◽  
N Terminus ◽  
Dynein Arms

Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multisubunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that the IFT46 N-terminus, ODA16, and outer arm dynein interact for IFT of the latter.

scholarly journals Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Hongki Song ◽  
Thomas L Torng ◽  
Amy S Orr ◽  
Axel T Brunger ◽  
William T Wickner
Keyword(s):  
Amino Acids ◽  
Membrane Fusion ◽  
Central Region ◽  
Coiled Coils ◽  
C Terminus ◽  
N Terminus ◽  
Oligomeric Assembly ◽  
Polar Residues

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/aSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

Kinetic Analysis of the Interactions Between the Different ELL and EAF Proteins Involved in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3116-3116
Author(s):  
Nimisha Sharma ◽  
Elena Solomaha ◽  
Federico Simone ◽  
Michael Thirman
Keyword(s):  
Amino Acids ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Full Length ◽  
Fusion Partner ◽  
Transcriptional Elongation ◽  
C Terminus ◽  
N Terminus ◽  
Acute Myeloid

Abstract The ELL gene was first cloned as a fusion partner of MLL in the (11;19)(q23;p13.1) translocation that occurs in acute myeloid leukemia. Subsequently, the ELL2 gene was cloned on the basis of its sequence homology to ELL. Both proteins stimulate the rate of transcript elongation by RNA polymerase II. Previously, we isolated two closely related proteins, EAF1 and EAF2, which interact with ELL and ELL2. Deletion mapping studies carried out to delineate the domain(s) of ELL involved in its interaction with either EAF1 or EAF2 showed that the N-terminus (amino acids 1–207) of ELL binds to both EAF1 and EAF2. In comparison, the middle region (207–411 amino acids) does not bind to either of the two EAF proteins and the C-terminus region (411–621 amino acids) binds only to the EAF1 protein. Biochemical studies have revealed that EAF1 and EAF2 enhance the rate of mRNA chain elongation by the ELL proteins in vitro. Although both ELL and ELL2 have similar roles in transcriptional elongation, ELL2 has not been shown to be involved in any hematological abnormality so far. In an attempt to gain a deeper understanding of the biology and functions of the interactions between these different proteins, we determined the kinetic properties of these interactions using the biophysical techniques of surface plasmon resonance (SPR) and isothermal calorimetry (ITC). SPR detects complex formation in real time and provides a better comprehension of the dynamics of association and dissociation of an interaction, and ITC is used to determine the thermodynamics of the interaction. Our SPR analysis has provided novel insights into the nature of the binding of the ELL proteins to the EAF proteins. We observed that both ELL and ELL2 bind to EAF1 and EAF2 with a high affinity, but the binding affinity of ELL2 for both EAF1 and EAF2 is almost twelve-fold greater than the affinity of ELL for both the EAF proteins. The higher affinity of ELL2 is due to much slower uptake and release kinetics reflected by the low association and dissociation rate constants of ELL2 compared to ELL. The stoichiometry of ELL, ELL2, EAF1 and EAF2 in the ELL-EAF1, ELL-EAF2, ELL2-EAF1 and ELL2-EAF2 complexes was estimated to be 1:1 after fitting the respective sensorgrams obtained by SPR analysis to the Langmuir’s bimolecular model. Interestingly, we did not observe any difference in the affinity of either ELL or ELL2 for binding to EAF1 or EAF2. We used SPR-based competition experiments to show that ELL and ELL2 bind to the same sites on the EAF proteins. We have also investigated the characteristics of binding of the various ELL domains to the EAF1/2 proteins. In the (11;19)(q23;p13.1) translocation, the C-terminus of ELL fuses to the N-terminus of MLL to generate a chimeric protein that interacts with EAF1 and this interaction is critical for the role of ELL in cell immortalization in vitro and leukemogenesis in vivo. In agreement with this observation, we found that the C-terminus of ELL binds EAF1 with a higher affinity than EAF2, while the N-terminus of ELL binds with similar affinities and displays similar kinetics of binding to both EAF1 and EAF2. We also found that the individual binding sites on the ELL N-terminus and the C-terminus exhibited a lower affinity for the EAF proteins, but the affinity increases when the two sites function together in the context of the full-length protein, suggesting that the two sites co-operate with each other to increase the affinity for the full-length ELL protein. Taken together, these observations suggest that although ELL and ELL2 share many similarities in terms of their sequence and function in transcription elongation, they bind to the EAF proteins with different affinities and kinetics. Alternative interaction dynamics and the interplay between the different ELL and EAF proteins permit distinct functional regulation of transcriptional elongation in normal and leukemic cells.

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