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Technology Topics | Liquid-Liquid Phase Separation (LLPS): A New Frontier in Modern Biomedicine]

2024-08-04

Liquid-liquid phase separation (Liquid-Liquid Phase Separation, LLPS) refers to the formation of phase separation droplets with different components and properties through the interaction of certain biological macromolecules (such as proteins and RNA) in cells [1]. These droplets are similar to the isolated state of oil droplets in water, forming a unique substructure within the cell. The phenomenon of liquid-liquid phase separation is widespread in the nucleus, cytoplasm and organelles, and is an important part of many cellular processes.

Biological Function of 1. Liquid-liquid Phase Separation
1. Formation of membraneless organelles
LLPS is a fundamental mechanism for the formation of membraneless organelles (membraneless organelles). These membraneless organelles include nucleoli, pressure granules, Cajal bodies, P-bodies, etc. They are not surrounded by a lipid bilayer membrane, but are capable of bringing together specific proteins and nucleic acid molecules by the mechanism of LLPS.
2. Dynamic regulation and rapid response
LLPS allows cells to rapidly and reversibly regulate the aggregation and dispersion of proteins and RNA, which is a key mechanism for cells to respond to environmental changes and signal transduction [2].
3. Regulation of gene expression
LLPS plays an important role in the regulation of gene expression. In the process of RNA processing, some membraneless organelles formed by LLPS are rich in proteins and factors related to RNA processing, which can focus on the key molecules in the process of RNA editing, splicing and degradation. During translation, LLPS is able to regulate the expression of specific mRNAs at the translational level by forming microdomains of translational inhibition or activation.
4. Metabolic regulation
Certain reactions in cellular metabolism need to be organized and optimized by the LLPS mechanism.
5. Disease Occurrence
Abnormalities in LLPS are often associated with disease, particularly in diseases associated with protein aggregation, such as neurodegenerative diseases (Alzheimer's disease, Parkinson's disease) and certain types of cancer.

2.Study on phase separation in tumor

1. Phase separation regulates gene expression
Researchers found that among the m6A reading proteins, the nuclear m6A reading protein YTHDC1 is particularly critical for AML cell survival [3]. YTHDC1 is significantly highly expressed in multiple different subtypes of AML case samples. By knocking down YTHDC1, it was found that AML cell proliferation was slowed down, myeloid differentiation was enhanced, apoptosis was increased and the development of AML in mice was significantly inhibited. This study reveals the YTHDC1 binding m6A-mRNA through the way of phase separation to form nuclear condensation, protect the target gene is not degraded, promote gene expression mechanism.
1) in AML cell lines shRNA knockdown/CRISPR knockdown YTHDC1This leads to slower proliferation of AML cells, enhanced myeloid differentiation and increased apoptosis.

2)CRISPR/Cas9 gene knock-in for endogenous YTHDC1 plus EGFP tagIt is proved that YTHDC1 protein is separated by liquid-liquid phase in vivo and in vitro.

3)Point mutation or fragment knockoutCharacterization of the IDR domain in YTHDC1 is critical for LLPS in cells.

 

Products and services related to "phase separation research:

1. Gene knockout customization service: Large fragment/precise/non-precise knockout for the customer-specified area (coding area/non-coding area);

2. Inducible shRNA knockdown stable cell lines: For essential genes, we provide inducible knockdown stable cell line protocols;

3. Construction of stable cell lines by in situ insertion of gene taggingBased on dTAG precise targeting protein degradation, real-time monitoring of protein function;

4. Construction of stable cell lines by in situ insertion of gene tagging: EGFP,RFP,Strep-tag and other tags are inserted into the N-terminal/C-terminal of the gene in situ;

5. Construction of point mutation stable cell lineSite-directed mutagenesis of the amino acids of the gene specified by the customer to construct a stable cell line.

 

2. Phase separation is involved in DNA damage and repair

Researchers have revealed a new mechanism for RAP80-mediated recruitment of BRCA1 [4], providing new insights into the role of phase separation in DNA double-strand break repair. The binding of RAP80 to the Lys63-linked polyubiquitin chain increases the multivalent interaction, thereby inducing the liquid-liquid phase separation (LLPS) of RAP80 at the site of DNA damage. At the same time, it was found that RAP80 LLPS enhanced the radiation resistance of tumor cells, indicating that RAP80 may be a potential target for tumor radiotherapy.

The study was adoptedCRISPR Knockout RAP80It is proved that it forms a liquid condensate in the nucleus, and the elimination of RAP80 agglutination significantly inhibits the formation of BRCA1 focus, revealing the key role of RAP80 agglutination in BRCA1 recruitment and radiation sensitivity.

3. Phase separation regulates tumorigenesis
Studies have found that circular RNA circVAMP3 inhibit the occurrence and development of hepatocellular carcinoma (HCC) by promoting the separation of CAPRIN1 proteins [5] and forming stress granules in cells to prevent the translation of c-Myc mRNA.

1) through gene editing inknockdown circVAMP3 in cells and miceIt is proved that it can inhibit the proliferation and migration of HCC cells.

2) The study found that circVAMP3 recruited CAPRIN1-G3BP1 complexes, resulting in protein aggregation and phase separation, promoting the formation of stress particles in cells; Knock down circVAMP3Stress particles decreased.

4. Phase separation and tumor immunity
The study found that under the stimulation of IFN γ, KAT8-IRF1 in tumor cells can enhance the molecular mechanism of PD-L1 expression by forming aggregates with the function of promoting transcription [6]. It was found that the formation of aggregates can enhance the catalytic rate of KAT8 acetylation of IRF1. A blocking polypeptide that inhibits the formation of aggregates was developed and its antitumor activity was proved.
1) Researchers use whole genome CRISPR-Cas9 libraryscreening, found that KAT8 can regulate the expression of PD-L1 gene knockoutValidated in cells.

2) T urboID Proximity Labeling Combined Mass SpectrometryThe researchers identified an interaction between the transcription factor IRF1 and KAT8.

3) in KAT8 N-terminal insert mEGFP, IRF1C end insert mCherry tagDetection of the two localization, found that the two co-expression in the nucleus can form a droplet-like condensation.

4) UtilizationdCas9-SunTag-sgARRAYConducting genomic mapping, the researchers observed that endogenous KAT8-IRF1 aggregates can be localized to the PD-L1 promoter.

 

Products and services related to "phase separation research:

1. Whole genome gene knockout mixed pooled library screening:Greater throughput and less time-consuming, suitable for phenotypes that can be changed by selective pressure, such as cell viability/proliferation assays or FACS analysis;

2. Screening of human/mouse gene knockout array arrayed library: For 19 metabolic pathways, higher accuracy, simplified data interpretation, strong experimental repeatability, and broader research needs;

3. Construction of stable cell lines by in situ insertion of gene tagging：Gene in situ N-terminal/C-terminal insertion of EGFP,mCherry and other tags for co-localization studies;

4. Construction of stable cell lines by in situ insertion of gene tagging：The gene is inserted into birID at the N-terminal/C-terminal in situ, and TurboID and other tags are used for adjacent labeling detection, which can find protein-protein interaction (weak interaction, transient interaction); Strep-tag and other tags are inserted in situ for AP-MS detection, which can find strong protein-protein interaction;

5. Granular manstrep II purified packingVery high affinity enrichment (100-fold higher than antibody enrichment) for Strep-tag proteins as well as biotin-labeled proteins.

6. AP-MS/IP-MS detection: Mass spectrometry detection of cell samples adjacent to the label or affinity label (Strep-tag) to find interacting proteins;

7. Construction of stable cell lines by in situ insertion of gene tagging：Live cell imaging analysis of genomic loci dCas9-SunTag the system and fluorescent proteins.

 

The importance of 3. gene editing technology in phase separation research

Gene editing at the cellular level in vitro (knock-out/knock-in) has become a regular technique in phase separation studies. Biological studies on phase separation, as well as in cancer, have played a key role. With the further development of phase separation research, it will also lead to the further integration of gene editing technology in this field.

As a technology-driven company focusing on the development of high-throughput gene editing tools, Graneman Bio will continue to focus on this field and launch related products and services to comprehensively assist researchers in phase separation research!

References

[1] Razin, S., Gavrilov, A.The Role of Liquid–Liquid Phase Separation in the Compartmentalization of Cell Nucleus and Spatial Genome Organization. Biochemistry Moscow 85, 643–650 (2020).
[2] Alberti S, Gladfelter A,et al.Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.Cell. 2019 Jan 24;176(3):419-434.
[3] Cheng Y, Xie W, et al.N6-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation. Cancer Cell. 2021 Jul 12;39(7):958-972.e8.
[4] Qin C, Wang YL, et al.RAP80 phase separation at DNA double-strand break promotes BRCA1 recruitment. Nucleic Acids Res. 2023 Oct 13;51(18):9733-9747.
[5] Chen S, Cao X, et al.circVAMP3 Drives CAPRIN1 Phase Separation and Inhibits Hepatocellular Carcinoma by Suppressing c-Myc Translation. Adv Sci (Weinh). 2022 Mar;9(8):e2103817.
[6] Wu Y, Zhou L, et al.Disrupting the phase separation of KAT8-IRF1 diminishes PD-L1 expression and promotes antitumor immunity. Nat Cancer. 2023 Mar;4(3):382-400.
 
 

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