pH-Induced Folding of the Caspase-Cleaved Par-4 Tumor Suppressor: Evidence of Structure Outside of the Coiled Coil Domain

Clark, Andrea M.; Ponniah, Komala; Warden, Meghan S.; Raitt, Emily M.; Yawn, Andrea; Pascal, Stephen M.

doi:10.3390/biom8040162

Cited by 7 publications

(16 citation statements)

References 94 publications

(153 reference statements)

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“…This region of the protein can be further subdivided as follows: ( Figure 1 a): the SAC (Selective for Apoptosis induction in Cancer cells) domain, the CC (Coiled-Coil) domain, and the Linker between these two domains. In addition, the SAC domain contains an NLS (Nuclear Localization Signal) near its N-terminus, and the CC domain contains an LZ (Leucine Zipper) domain which comprises its C-terminal half [ 23 ]. Prediction of disorder in these domains was done by using DISOPRED3, which utilizes X-ray diffraction databases ( Figure 1 b) [ 27 , 28 ].…”

Section: Resultsmentioning

confidence: 99%

“…Considering a probability of 0.5 (dashed line) as the demarcation line, the analysis clearly predicts order in the CC domain and disorder in the Linker, while the SAC domain has intermediate character, approaching the order/disorder line. Previous sequence analysis also showed mixed order/disorder propensity and some helical character in the SAC domain, and so order in the SAC domain would not be surprising, under the right conditions [ 23 ].…”

Section: Resultsmentioning

confidence: 99%

“…We have previously shown that high levels of sodium chloride or acidic pH can induce the cl-Par-4 fragment to form highly compact, highly helical structures in vitro, at high micromolar protein concentrations [ 23 , 24 ]. The ionic conditions required to favor these small, well-folded particles are outside of the range of conditions normally found in cells.…”

Section: Introductionmentioning

confidence: 99%

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Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment

2021

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Prostate apoptosis response-4 (Par-4) is a proapoptotic tumor suppressor protein that has been linked to a large number of cancers. This 38 kilodalton (kDa) protein has been shown to be predominantly intrinsically disordered in vitro. In vivo, Par-4 is cleaved by caspase-3 at Asp-131 to generate the 25 kDa functionally active cleaved Par-4 protein (cl-Par-4) that inhibits NF-κB-mediated cell survival pathways and causes selective apoptosis in tumor cells. Here, we have employed circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) to assess the effects of various monovalent and divalent salts upon the conformation of cl-Par-4 in vitro. We have previously shown that high levels of sodium can induce the cl-Par-4 fragment to form highly compact, highly helical tetramers in vitro. Spectral characteristics suggest that most or at least much of the helical content in these tetramers are non-coiled coils. Here, we have shown that potassium produces a similar effect as was previously reported for sodium and that magnesium salts also produce a similar conformation effect, but at an approximately five times lower ionic concentration. We have also shown that anion identity has far less influence than does cation identity. The degree of helicity induced by each of these salts suggests that the “Selective for Apoptosis in Cancer cells” (SAC) domain—the region of Par-4 that is most indispensable for its apoptotic function—is likely to be helical in cl-Par-4 under the studied high salt conditions. Furthermore, we have shown that under medium-strength ionic conditions, a combination of high molecular weight aggregates and smaller particles form and that the smaller particles are also highly helical, resembling at least in secondary structure, the tetramers found at high salt.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment

2021

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“…Protein expression and purification was performed according to published procedures . Briefly, cl‐Par‐4 in the H‐MBP‐3C expression vector was expressed in BL21(DE3) CodonPlus Escherichia coli grown in LB with 100 μg·mL −1 ampicillin at 37 °C, and purified using a His‐Trap HP column (GE Healthcare, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

“…Previous in vitro studies have shown that cl‐Par‐4 is predominantly disordered under typical physiological conditions, but that acidic pH promotes increased helical content . Disordered proteins often fold under extreme conditions due to various stabilizing factors .…”

Section: Introductionmentioning

confidence: 99%

Tetramer formation by the caspase‐activated fragment of the Par‐4 tumor suppressor

et al. 2019

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The prostate apoptosis response‐4 (Par‐4) tumor suppressor can selectively kill cancer cells via apoptosis while leaving healthy cells unharmed. Full length Par‐4 has been shown to be predominantly intrinsically disordered in vitro under neutral conditions. As part of the apoptotic process, cellular Par‐4 is cleaved at D131 by caspase‐3, which generates a 24 kDa C‐terminal activated fragment (cl‐Par‐4) that enters the nucleus and inhibits pro‐survival genes, thereby preventing cancer cell proliferation. Here, the structure of cl‐Par‐4 was investigated using CD spectroscopy, dynamic light scattering, intrinsic tyrosine fluorescence, and size exclusion chromatography with mutli‐angle light scattering. Biophysical characterization shows that cl‐Par‐4 aggregates and is disordered at low ionic strength. However, with increasing ionic strength, cl‐Par‐4 becomes progressively more helical and less aggregated, ultimately forming largely ordered tetramers at high NaCl concentration. These results, together with previous results showing induced folding at acidic pH, suggest that the in vivo structure and self‐association state of cl‐Par‐4 may be strongly dependent upon cellular environment.

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Evidence of direct interaction between cisplatin and the caspase‐cleaved prostate apoptosis response‐4 tumor suppressor

Raut,

Pandey,

Kharel

et al. 2024

Protein Science

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Prostate apoptosis response‐4 (Par‐4) tumor suppressor protein has gained attention as a potential therapeutic target owing to its unique ability to selectively induce apoptosis in cancer cells, sensitize them to chemotherapy and radiotherapy, and mitigate drug resistance. It has recently been reported that Par‐4 interacts synergistically with cisplatin, a widely used anticancer drug. However, the mechanistic details underlying this relationship remain elusive. In this investigation, we employed an array of biophysical techniques, including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and UV‐vis absorption spectroscopy, to characterize the interaction between the active caspase‐cleaved Par‐4 fragment (cl‐Par‐4) and cisplatin. Additionally, elemental analysis was conducted to quantitatively assess the binding of cisplatin to the protein, utilizing inductively coupled plasma‐optical emission spectroscopy (ICP‐OES) and atomic absorption spectroscopy (AAS). Our findings provide evidence of direct interaction between cl‐Par‐4 and cisplatin, and reveal a binding stoichiometry of 1:1. This result provides insights that could be useful in enhancing the efficacy of cisplatin‐based and tumor‐suppressor‐based cancer therapies.This article is protected by copyright. All rights reserved.

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pH-Induced Folding of the Caspase-Cleaved Par-4 Tumor Suppressor: Evidence of Structure Outside of the Coiled Coil Domain

Cited by 7 publications

References 94 publications

Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment

Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment

Tetramer formation by the caspase‐activated fragment of the Par‐4 tumor suppressor

Evidence of direct interaction between cisplatin and the caspase‐cleaved prostate apoptosis response‐4 tumor suppressor

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