Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH

Hlodan, Roman; Pain, Roger H.

doi:10.1016/0014-5793(94)80567-9

Cited by 38 publications

(34 citation statements)

References 23 publications

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“…The kinetics of folding from this state to the native state have beeh measured and shown to be first order, with the same rate constant as that for the transition followed at neutral pH from the state fully unfolded in denaturant (Hlodan and Pain, 1994). This indicates that the rate-limiting step in folding to the native state occurs from the same intermediate species in each case, showing that TNF folds through a compact, trimeric molten globule intermediate.…”

Section: Discussionmentioning

confidence: 68%

“…At low pH TNF has been shown to exhibit the properties of a compact, trimeric molten globule (Hlodan and Pain, 1994). The kinetics of folding from this state to the native state have beeh measured and shown to be first order, with the same rate constant as that for the transition followed at neutral pH from the state fully unfolded in denaturant (Hlodan and Pain, 1994).…”

Section: Discussionmentioning

confidence: 98%

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The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

Hlodan

Pain

1995

European Journal of Biochemistry

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The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor-a (TNF) were studied by chemical cross-linking. This revealed the rapid accumulation of a dimeric interrnediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8-anilino-I-naphthalene sulfonic acid fluorescence are first order and, combined with the kinetics of association, reveal the presence of folding steps both before and subsequent to formation of the trimer. Results from gel exclusion chromatography and kinetics, together with the existence of an acid-induced molten globule, support the conclusion that TNF folds and assembles through a trimeric molten globule.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 98%

The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

Hlodan

Pain

1995

European Journal of Biochemistry

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“…The finding that a molten globule intermediate can undergo oligomerization comparable to the native state is not surprising, however. It has already been reported for tumour necrosis factor [49] that both the native and molten globule states are trimeric.…”

Section: Discussionmentioning

confidence: 86%

Characterization of the Equilibrium Intermediates in Acid Denaturation of Human Stefin B

Žerovnik

Jerala

Kroon-Žitko

et al. 1997

European Journal of Biochemistry

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Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCI) four states can be distinguished: N -1, -I, -U, where N is the native state, I, is a native-like intermediate, I, is an acid intermediate state with properties of a molten globule and U is the unfolded state. State I, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1 -aniline-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is threestate between the dimeric native state N, and intermediates I,, and I,, which are also dimeric according to size-exclusion chromatography. The acid intermediate I, is more structured than I, : it binds ANS to a lower extent an I,, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 'H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18°C): state 1, from pH 5.0 to 4.6, 0.03 M salt; state I, below pH 3.8, 0.42 M salt; and state I, in equilibrium with I, at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I, and I, were comparable to those of the native state. The enthalpy of unfolding for state I, could not be determined. . . ~ ing. The enthalpy of unfolding of the a-lactalbumin molten globule could be obtained indirectly by deconvolution of the thermograms under conditions where the molten globule and the native states were in equilibrium (161. A direct measurement of the enthalpy of unfolding was described for the acid molten globule state of apo-myoglobin, stabilized by 250 mM salt [17]. KeywordsDifferent proteins behave very differently on acid denaturation [18]. Some do not unfold at the lowest pH values, some undergo transition to a compact, molten globule state A, and a third type first unfolds to an extended conformation and then undergoes transition to a compact state A on addition of anions [19, 201. Intermediates of the molten globule type have been detected in acid denaturation for a number of proteins [21-281. Human stefin B, a globular protein without disulfide bonds, is a member of the stefin family of cysteine proteinase inhibitors [29]. Its three-dimensional crystal structure in complex with papain [30] and the solution structure of the homologous protein human stefin A have been determined [31]. The 98-residue globular protein is folded in a P-pleated-sheet structure, consisting of four longer and one shorter antiparallel strands onto which an 18-residue a-helix leans. Its denaturation and folding have been studied revealing equilibrium intermediates under several denaturing conditions [32...

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“…A similar effect to that of the anionic oligosaccharides might be exhibited by the anionic phospholipid containing membranes, which have been shown to shift induction of molten globule formation to higher pH (64,65). Molten globule conformation has been implicated in physiological processes such as membrane insertion and pore formation (36,66,67) or chaperone binding (68) and acid activation of the proteases may be another role of this type of protein conformation.…”

Section: Discussionmentioning

confidence: 99%

pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

Jerala

Žerovnik

Kidrič

et al. 1998

Journal of Biological Chemistry

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Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.All lysosomal and most other proteases are synthesized in the form of inactive precursors (1, 2). Propeptides are generally located N-terminal to the mature enzyme, and activation of the enzyme is accomplished by cis-or trans-cleavage of the propeptide. Propeptides vary from a few (e.g. trypsin) to more than 200 residues (e.g. cathepsin C). Longer propeptides are generally strong and specific inhibitors of their mature enzymes (3-7). In most cases propeptides are also indispensable for correct folding of the enzymes (8). In some enzymes folding of the mature form is extremely slow, and the propeptide assists in overcoming the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10). Proenzymes are quite often more stable than mature enzymes (11, 12) and can represent a pool of latent enzyme until the activation occurs in the proper conditions. Propeptides are also involved in targeting to specific organelles (13, 14); they can affect posttranslational modification such as glycosylation (15) and mediate interactions with other molecules (16,17). Propeptides can be cleaved either by other proteases or by intra-or intermolecular autocatalysis. pH change is one of the most common environmental parameters responsible for triggering the activation of proteases, occurring in cysteine, aspartic acid, and metalloproteases (1,18,19). Low pH is thought either to increase the susceptibility of the propeptide as a substrate due to the protonation of groups close to the cleavage site or to cause a conformational change in the propeptide or enzyme.Cathepsin L is one of the most active cysteine proteases and accounts for most of the lysosomal cysteine protease activity (20). It has been implicated in a range of processes incl...

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Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH

Cited by 38 publications

References 23 publications

The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein

Characterization of the Equilibrium Intermediates in Acid Denaturation of Human Stefin B

pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

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