The αlytic protease pro-region does not require a physical linkage to activate the protease domain in vivo

Silen, Joy L.; Agard, David A.

doi:10.1038/341462a0

Cited by 243 publications

(137 citation statements)

References 18 publications

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“…Expression of the mature portion of the protease in E. coli results in little or no activity [38]. Co-expression of the proregion in trans is required in order to reach the native form in viva, indicating that the proregion could play an essential role in folding [39]. This hypothesis has recently been confirmed with in vitro dcnaturation-renaturation experiments [40].…”

Section: a Proregion Kinetically Required For The Folding Of Alphasupporting

confidence: 62%

Amino acid substitutions influencing intracellular protein folding pathways

Mitraki

King

1992

FEBS Letters

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Though an increasing variety of chaperonins areemerging as important factors in directing polypeptidc chain folding off the ribosome, the primary amino acid sequence remains the major determinant of final conformation. The ability to identify cytoplasmic folding intermediates in the formation of the tailspike cndorhamnosidase of phage P22 has made it possible to isolate two classes of mutations influencing folding intermediatestemperature-sensitive folding mutalions and global suppressors of r!fmutants. These and related amino acid substitutiow in eukaryotic proteins arc discussed in the context of inclusion body formation and problems in the recovery of correctly folded proteins.Protein folding in vivo; Global suppressors; Pbagc P 22 tailspike; Inclusion body THE IMPORTANCE OF INTRACELLULAR FOLDING INTERMEDIATESThe nature of the grammar through which amino acid sequences of polypeptide chains direct their threedimensional fold remains an unsolved problem in mo-!.ecular biology. A great deal of effort has focused on identifying residues and sequences which contribute to the stability and activity of the native state (for reviews, see [l-4]). However, investigations of the actual pathways through which newly synthesized polypeptide chains reach their native state reveals that considerable sequence information is necessary for folding into the native state, in addition to the information required to stay in that state once achieved. Within the aqueous environment of the cytoplasm polypeptide chains do not fold through two-state transitions but pass through well-defined, though poorly understood, intermediate states [S-S]. These folding intermediates have to solve a variety of problems: avoiding aggregated states; avoiding proteolysis; reaching particular cellular compartments; transiently binding chaperonks and other auxiliary proteins; and finally, reaching the native state, In fact folding intermediates generally have properties quite distinct from their own native states, in terms of solubility, stability, half-lives and interactions with cellular components, such as chaperonins or signal recognition particles. As a result there Corwqx~~~lr,~cr ucldrew A. Mitraki.

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Section: a Proregion Kinetically Required For The Folding Of Alphasupporting

confidence: 62%

Amino acid substitutions influencing intracellular protein folding pathways

Mitraki

King

1992

FEBS Letters

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“…However, there are instances in which a few proteins, such as subtilisin E [2], α-lytic protease [3], and carboxypeptidase Y [4], could not return to their natively folded conformation from their denatured state despite the removal of the denaturant, indicating that the refolding/unfolding processes of those proteins were not reversible. These observations have raised questions about the validity of earlier conclusion from the unfolding and refolding experiments of ribonuclease.…”

Section: Introductionmentioning

confidence: 99%

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

et al. 2008

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Equilibrium unfolding of a 69-kDa monomeric Escherichia coli maltodextrin glucosidase (MalZ) was studied using intrinsic and extrinsic fluorescence spectroscopy. The unfolding transition of MalZ followed a three-state process, involving the formation of a stable intermediate state having more exposed hydrophobic surface. It was found that the protein structure can be easily perturbed by low concentration of guanidium hydrochloride (GdnHCl) and, at a GdnHCl concentration of 2 M, MalZ was denatured completely. The active site of the protein also has been proved to be sensitive to a low concentration of GdnHCl since MalZ deactivated at 0.5 M GdnHCl completely. The surface hydrophobicity and ANS-binding site of the protein have been determined to be 150.7 and 0.24, respectively. Perhaps the formation of the stable unfolding intermediate, having higher surface hydrophobicity, may be one of the reasons for aggregation of MalZ and its recognition by chaperonin GroEL during the assisted folding pathway.

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“…(a) As a chaperonin in analogy to pro-peptides of prokaryotic serine proteinases. In prokaryotes, pro-sequences are necessary for guiding the folding process of serine proteinases into an active conformation [50,511. Separate expression of pro-PrB and superpeptide might allow to test this model.…”

Section: Discussionmentioning

confidence: 99%

“…Since the serine to alanine replacement already blocks processing of the super-pro-PrB, separate expression of superpeptide and pro-PrB, analogously to the one described for the a-lytic protease [50], have to be carried out. Because of the high number of charged residues, doubts have been raised as to whether the superpeptide is able to translocate through the lipid bilayer.…”

Section: Discussionmentioning

confidence: 99%

Biogenesis of the yeast vacuole (lysosome)

Hirsch

Schiffer

Müller

et al. 1992

European Journal of Biochemistry

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The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prhl-Ala519. Following replacement of the wildtype PRBl allele withprhl-AZu519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prbl-A11519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Eschrrichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDSjPAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wildtype cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wildtype cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin. In a more detailed kinetic analysis, rapid PrA-triggered degradation of superpeptide was observed only coincidently with the maturation of the PrA-processed 40-kDa intermediate pro-PrB form to the mature PrB of 33 kDa. Slow degradation of the superpeptide only by incubation was correlated with unmasking of the cryptic pro-PrB activity. These results suggest that the complex maturation events of the vacuolar serine PrB precursors involve several timely and spatially controlled (auto-)proteolytic proces...

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The αlytic protease pro-region does not require a physical linkage to activate the protease domain in vivo

Cited by 243 publications

References 18 publications

Amino acid substitutions influencing intracellular protein folding pathways

Amino acid substitutions influencing intracellular protein folding pathways

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

Biogenesis of the yeast vacuole (lysosome)

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