scholarly journals Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

2006 ◽  
Vol 5 (12) ◽  
pp. 2174-2183 ◽  
Author(s):  
Susannah D. Brydges ◽  
Xing Wang Zhou ◽  
My-Hang Huynh ◽  
Jill M. Harper ◽  
Jeffrey Mital ◽  
...  
Keyword(s):  
Cell Invasion ◽  
Limited Proteolysis ◽  
Opportunistic Pathogen ◽  
Proteolytic Processing ◽  
Secretory Proteins ◽  
Membrane Association ◽  
Cleavage Sites ◽  
Targeted Deletion ◽  
Genetic Ablation ◽  
Substrate Cleavage

ABSTRACT Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.

scholarly journals A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

Blood ◽  
2009 ◽  
Vol 113 (16) ◽  
pp. 3792-3800 ◽  
Author(s):  
John E. Weldon ◽  
Laiman Xiang ◽  
Oleg Chertov ◽  
Inger Margulies ◽  
Robert J. Kreitman ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Proteolytic Processing ◽  
Lymphocytic Leukemia ◽  
Cleavage Sites ◽  
Pseudomonas Exotoxin A ◽  
Lysosomal Protease ◽  
Tumor Associated Antigen ◽  
Protease Digestion ◽  
Higher Doses ◽  
Increased Activity

Abstract Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.

Proteolytic processing of secretory proteins in Paramecium: immunological and biochemical characterization of the precursors of trichocyst matrix proteins

1992 ◽  
Vol 103 (2) ◽  
pp. 349-361 ◽  
Author(s):  
S.J. Shih ◽  
D.L. Nelson
Keyword(s):  
Cross Sections ◽  
Disulfide Bonds ◽  
Biochemical Characterization ◽  
Rabbit Antiserum ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Proteolytic Processing ◽  
Matrix Proteins ◽  
Secretory Proteins

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precursor serum after preadsorption no longer stained the central, paracrystalline region, but still stained the peripheral as well as the structureless region of the secretory granule. In trichocyst-developing mutants tl (trichless) and ftA (football A), the precursors for all four groups of mature proteins were present but their processing was affected: severely blocked in tl (which has no recognizable crystalline trichocyst matrix), and partially blocked in ftA (which has some abnormal trichocyst matrices with crystalline centers). These observations constitute further evidence that proteolytic processing of precursors occurs in parallel with crystallization.

scholarly journals Proteolytic maturation of α2δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ivan Kadurin ◽  
Laurent Ferron ◽  
Simon W Rothwell ◽  
James O Meyer ◽  
Leon R Douglas ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Proteolytic Cleavage ◽  
Proteolytic Processing ◽  
Calcium Currents ◽  
Cleavage Sites ◽  
Voltage Dependent ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
Synaptic Boutons

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.

Transport and proteolytic processing of rabbit cardiac cathepsin D

1985 ◽  
Vol 248 (1) ◽  
pp. C135-C144
Author(s):  
A. M. Samarel ◽  
A. G. Ferguson ◽  
S. W. Worobec ◽  
M. Lesch
Keyword(s):  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteolytic Processing ◽  
Time Dependent ◽  
Low Density ◽  
Enzyme Maturation

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

scholarly journals Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites

1991 ◽  
Vol 202 (1) ◽  
pp. 48-51
Author(s):  
Kerstin G. KRIEGLSTEIN ◽  
Agnes H. HENSCHEN ◽  
Ulrich WELLER ◽  
Ernst HABERMANN
Keyword(s):  
Tetanus Toxin ◽  
Limited Proteolysis ◽  
Cleavage Sites

scholarly journals PROSPERous: high-throughput prediction of substrate cleavage sites for 90 proteases with improved accuracy

2017 ◽  
Vol 34 (4) ◽  
pp. 684-687 ◽  
Author(s):  
Jiangning Song ◽  
Fuyi Li ◽  
André Leier ◽  
Tatiana T Marquez-Lago ◽  
Tatsuya Akutsu ◽  
...  
Keyword(s):  
High Throughput ◽  
Cleavage Sites ◽  
Substrate Cleavage ◽  
Improved Accuracy

scholarly journals Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Srijana Upadhyay ◽  
Xinping Xu ◽  
Xiaorong Lin
Keyword(s):  
Secondary Metabolism ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Polyketide Synthases ◽  
Common Mechanism ◽  
Secretory Proteins ◽  
Membrane Association ◽  
Lipid Modification ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases

ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus . Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. IMPORTANCE Subcellular compartmentalization is increasingly recognized as an important aspect of fungal secondary metabolism. It facilitates sequential enzymatic reactions, provides mobility for enzymes and metabolites, and offers protection against self-toxification. However, how compartmentalization is achieved remains unclear given that the majority of enzymes encoded by secondary metabolism gene clusters are predicted to be cytosolic proteins. Through studying melanization in Aspergillus , we previously found that all enzymes involved in the early steps of melanization are atypical secretory proteins. Here, we discovered physical interactions among melanin enzymes. However, it was the posttranslational palmitoylation rather than the physical interaction that was responsible for their recruitment to the secretory pathway. Intriguingly, palmitoylation is likely a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus . Collectively, our findings suggest that posttranslational lipid modification helps direct secondary metabolism to defined organelles for biosynthesis and trafficking.

scholarly journals A Forward Genetic Strategy Reveals Destabilizing Mutations in the Ebolavirus Glycoprotein That Alter Its Protease Dependence during Cell Entry

2009 ◽  
Vol 84 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Anthony C. Wong ◽  
Rohini G. Sandesara ◽  
Nirupama Mulherkar ◽  
Sean P. Whelan ◽  
Kartik Chandran
Keyword(s):  
Proteolytic Processing ◽  
Cell Entry ◽  
Cleavage Sites ◽  
Viral Glycoprotein ◽  
Enveloped Virus ◽  
Cysteine Cathepsins ◽  
Vesicular Stomatitis ◽  
Lysosomal Proteases ◽  
Conformational Rearrangements ◽  
Viral Membrane Fusion

ABSTRACT Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the “clamp” that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.

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