Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid–Liquid Phase Separation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Retention of Involucrin in a Heat Lysate of Keratinocytes
2.2. Identification and Relationships among Keratinocyte-Expressed Proteins following Thermostability Fractionation
2.3. Intrinsic Disorder Traits of Thermostable Proteins Specific to or Highly Expressed in KCs
2.4. Intrinsic Disorder Traits of Broadly Expressed Proteins Retrieved in the KC Thermostable Lysate
2.5. Keratinocyte Proteins: Database Intrinsic Disorder Assessment
2.6. KC Cornified Envelope (CE) Components Are Candidates for LLPS
2.7. Conclusions
3. Materials and Methods
3.1. Cell Culture
3.2. Protein Preparation and Analysis
3.3. Liquid Chromatography (LC) Tandem Mass Spectrometry (MS, LC MS/MS)
3.4. Bioinformatics: Intrinsic Disorder, Sequence Retrieval, and Phase Separation Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Protein | UniProt # | AA | Protein Role | PONDR-FIT (0–1) | RAPID Disorder % | SLIDER IDR (0–1) | catGRAN a |
---|---|---|---|---|---|---|---|
Involucrin b | P07476 | 585 | CE component, cornification c | 0.812 | 65.81 | 0.917 | 0.821 |
Ladinin-1 | O00515 | 517 | ECM and cadherin binding | 0.774 | 68.28 | 0.929 | 0.333 |
BP180 | Q9UMD9 | 1497 | Transmembrane attachment to ECM | 0.736 | 40.75 | 0.913 | 3.184 |
Cornifin A d | P35321 | 89 | CE component, cornification | 0.551 | 79.78 | 0.848 | −2.527 |
AHNAK/Desmoyokin d | Q09666 | 5890 | Scaffolding, nuclear-cytoplasmic shuttling | 0.494 | 48.37 | 0.851 | 2.942 |
Periplakin | O60437 | 1756 | CE component, scaffolding | 0.491 | 30.30 | 0.903 | 0.857 |
Galectin-3 d | P17931 | 250 | Cell–cell, cell–ECM interaction, splicing | 0.501 | 18.00 | 0.487 | 1.571 |
Protein | UniProt # | AA | Protein Role | PONDR-FIT (0–1) | RAPID Disorder % | SLIDER IDR (0–1) | catGRAN a |
---|---|---|---|---|---|---|---|
Prelamin A/C | P02545 | 664 | Nuclear membrane support, scaffolding | 0.620 | 34.79 | 0.9015 | 0.904 |
TAB182 | Q9C0C2 | 1729 | Nuclear and cyto-plasmic RNA-binding | 0.734 | 51.13 | 0.9281 | 1.975 |
Calpastatin | P20810 | 708 | Calpain protease inhibitor | 0.804 | 78.39 | 0.9565 | 1.010 |
SERBP1 | Q8NC51 | 408 | mRNA stability, PML formation | 0.751 | 60.54 | 0.9049 | 2.829 |
Nucleolin 1 | P19338 | 710 | RNA-binding, pre-rRNA transcription | 0.633 | 62.39 | 0.9357 | 2.495 |
ADIRF | Q15847 | 76 | Transcription factor | 0.810 | 50.00 | 0.6675 | −0.330 |
Protein | UniProt # | AA | Kappa | Omega | FCR | NCPR | Hydropathy | Dis. Prom. | PR |
---|---|---|---|---|---|---|---|---|---|
Loricrin | P23490 | 312 | 0.252 | 1.787 | 0.026 | 0.019 | 4.244 | 0.827 | 1 |
Hornerin | Q86YZ3 | 2850 | 0.162 | 0.823 | 0.099 | 0.029 | 3.136 | 0.911 | 1 |
LC3EA | Q5TA76 | 89 | 0.309 | 0.420 | 0.124 | 0.079 | 3.602 | 0.775 | 1 |
SPR2A a | P35326 | 72 | 0.151 | 0.115 | 0.139 | 0.083 | 3.193 | 0.764 | 1 |
Filaggrin 2 | Q5D862 | 2391 | 0.227 | 0.621 | 0.144 | 0.003 | 3.134 | 0.883 | 1 |
KPRP a | Q5T749 | 579 | 0.185 | 0.172 | 0.174 | 0.036 | 3.711 | 0.710 | 1 |
Elafin | P19957 | 117 | 0.124 | 0.227 | 0.197 | 0.060 | 4.664 | 0.598 | 1 |
Filaggrin | P20930 | 4061 | 0.164 | 0.235 | 0.240 | 0.005 | 2.827 | 0.919 | 1 |
S10AB | P31949 | 105 | 0.149 | 0.213 | 0.248 | 0 | 4.160 | 0.619 | 1 |
Sciellin | O95171 | 688 | 0.143 | 0.134 | 0.285 | 0.035 | 3.455 | 0.657 | 2 |
Cystatin A | P01040 | 98 | 0.146 | 0.146 | 0.286 | −0.020 | 3.822 | 0.633 | 2 |
S10AA | P60903 | 97 | 0.098 | 0.231 | 0.289 | 0 | 4.139 | 0.598 | 2 |
Involucrin | P07476 | 585 | 0.144 | 0.060 | 0.294 | −0.130 | 2.996 | 0.776 | 2 |
Envoplakin | Q92817 | 2033 | 0.129 | 0.116 | 0.323 | −0.004 | 3.700 | 0.716 | 2 |
Periplakin | O60437 | 1756 | 0.138 | 0.086 | 0.362 | −0.031 | 3.518 | 0.695 | 3 |
Trichohyalin | Q07283 | 1943 | 0.116 | 0.110 | 0.581 | −0.021 | 1.995 | 0.838 | 3 |
Protein | UniProt # | AA | PONDR-FIT (0–1) | RAPID Disorder % | catGRAN. a | FuzDrop pLLPS (0–1) b | P Score c | DeePhase d | PSPred. d |
---|---|---|---|---|---|---|---|---|---|
Hornerin | Q86YZ3 | 2850 | 0.923 | 57.72 | 5.572 | 1.000 | 11.91 | 0.88 | 0.9527 |
Filaggrin 2 | Q5D862 | 2391 | 0.896 | 49.14 | 4.418 | 0.9999 | 12.03 | 0.87 | 0.9904 |
Filaggrin | P20930 | 4061 | 0.895 | 55.58 | 3.553 | 1.000 | 4.73 | 0.83 | 0.9908 |
Loricrin | P23490 | 312 | 0.840 | 62.82 | 7.808 | 0.9985 | 12.31 | 0.81 | 0.9958 |
Involucrin | P07476 | 585 | 0.812 | 65.81 | 0.821 | 0.9921 | 2.18 | 0.78 | 0.4945 |
Trichohyalin | Q07283 | 1943 | 0.753 | 73.60 | 1.312 | 0.9947 | 0.66 | 0.72 | 0.2190 |
LCE3A | Q5TA76 | 89 | 0.625 | 18.18 | −0.779 | 0.9978 | NA | 0.59 | 0.9596 |
Sciellin | O95171 | 688 | 0.620 | 41.57 | 1.306 | 0.9527 | 2.10 | 0.78 | 0.9546 |
KPRP | Q5T749 | 579 | 0.571 | 29.02 | −0.591 | 0.9867 | 5.72 | 0.82 | 0.9096 |
SPR2A | P35326 | 72 | 0.530 | 65.28 | −3.342 | 0.9868 | NA | 0.58 | 0.7971 |
Periplakin | O60437 | 1756 | 0.491 | 30.30 | 0.857 | 0.6168 | 0.38 | 0.59 | 0.2852 |
Envoplakin | Q92817 | 2033 | 0.478 | 26.41 | 0.780 | 0.8022 | 2.00 | 0.81 | 0.6487 |
S100-A11 | P31949 | 105 | 0.374 | 17.14 | −0.481 | 0.1275 | NA | 0.11 | 0.0125 |
Cystatin A | P01040 | 98 | 0.363 | 27.55 | 0.410 | 0.1673 | NA | 0.10 | 0.0050 |
Elafin | P19957 | 117 | 0.350 | 30.77 | −0.180 | 0.2376 | NA | 0.17 | 0.0574 |
S100-A10 | P60903 | 97 | 0.336 | 16.49 | −0.373 | 0.2124 | NA | 0.08 | 0.0033 |
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Samulevich, M.L.; Shamilov, R.; Aneskievich, B.J. Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid–Liquid Phase Separation. Int. J. Mol. Sci. 2022, 23, 14323. https://doi.org/10.3390/ijms232214323
Samulevich ML, Shamilov R, Aneskievich BJ. Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid–Liquid Phase Separation. International Journal of Molecular Sciences. 2022; 23(22):14323. https://doi.org/10.3390/ijms232214323
Chicago/Turabian StyleSamulevich, Michael L., Rambon Shamilov, and Brian J. Aneskievich. 2022. "Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid–Liquid Phase Separation" International Journal of Molecular Sciences 23, no. 22: 14323. https://doi.org/10.3390/ijms232214323