Enrichment of autophagic cargo by liquid–liquid phase separation allows the formation of distinct entities of macromolecular complexes to be degraded by selective autophagy.
Display omitted
•In this ...review we discuss the degradation of different macromolecular complexes by selective autophagy.•We discuss what it takes for these large macromolecular complexes to be degraded and point to future work.•We discuss the role of liquid–liquid phase separation in the enrichment of macromolecular complexes prior to selective engulfment by autophagy.
Proteins are known to perform an astonishing array of functions thanks to their ability to cooperate and modulate each other’s properties. Inside cells, proteins can assemble into large multi-subunit complexes to carry out complex cellular functions. The correct assembly and maintenance of the functional state of macromolecular protein complexes is crucial for human health. Failure to do so leads to loss of function and potential accumulation of harmful materials, which is associated with a variety of human diseases such as neurodegeneration and cancer. Autophagy engulfs cytosolic material in autophagosomes, and therefore is best suited to eliminate intact macromolecular complexes without disassembling them, which could interfere with de novo assembly. In this review, we discuss the role of autophagy in the selective degradation of macromolecular complexes. We highlight the current state of knowledge for different macromolecular complexes and their selective autophagic degradation. We emphasize the gaps in our understanding of what it takes for these large macromolecular complexes to be degraded and point to future work that may shed light on the regulation of the selective degradation of macromolecular complexes by autophagy.
Display omitted
•Biophysical principles of RSV virus factory assembly and modulation need to be elucidated.•RSV-P is the scaffold for biomolecular condensation both in vitro and in cells.•RSV-P ...restores nucleoprotein aggregates into liquid droplets.•RSV-P may act as the “solvent protein” within the viral factories.
Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid–liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a “solvent-protein”.
PML nuclear body (NB) malfunction often leads to acute leukemia outbreaks and other severe diseases. PML NB rescue is the molecular basis of arsenic success in acute promyelocytic leukemia (APL) ...treatment. However, it is unclear how PML NBs are assembled. Here, we observed the presence of liquid–liquid phase separation (LLPS) in NB formation by fluorescence recovery after photobleaching (FRAP) experiment. Compared with the wild‐type (WT) NBs, PML A216V derived from arsenic‐resistant leukemia patients markedly crippled LLPS, but not altered the overall structure and PML RBCC oligomerization. In parallel, we also reported several Leu to Pro mutations that were critical to PML coiled‐coil domain. FRAP characterization and comparison between L268P and A216V revealed markedly different LLPS activities in these mutant NBs. Transmission electron microscopy (TEM) inspections of LLPS‐crippled and uncrippled NBs showed aggregation‐ and ring‐like PML packing in A216V and WT/L268P NBs, respectively. More importantly, the correct LLPS‐driven NB formation was the prerequisite for partner recruitment, post‐translational modifications (PTMs), and PML‐driven cellular regulations, such as ROS stress control, mitochondria production, and PML‐p53‐mediated senescence and apoptosis. Altogether, our results helped to define a critical LLPS step in PML NB biogenesis.
Working hypothesis of LLPS‐driven PML NB biogenesis. In the first step, PML–PML interaction may be mainly mediated by RBCC oligomerization. In the second step, PML multimers can come together through LLPS, facilitated by local protein concentrations, PTMs, and recruitment of partner proteins, ultimately giving rise to a remarkable subcellular organelle with flexible sizes.
Long noncoding RNAs (lncRNAs), which are among the most well-characterized noncoding RNAs, have attracted much attention due to their regulatory functions and potential therapeutic options in many ...types of disease. Liquid–liquid phase separation (LLPS), the formation of droplet condensates, is involved in various cellular processes, but the molecular interactions of lncRNAs in LLPS are unclear. In this review, we describe the research development on LLPS, including descriptions of various methods established to identify LLPS, summarize the physiological and pathological functions of LLPS, identify the molecular interactions of lncRNAs in LLPS, and present the potential applications of leveraging LLPS in the clinic. The aim of this review is to update the knowledge on the association between LLPS and lncRNAs, which might provide a new direction for the treatment of LLPS-mediated disease.
•Liquid–liquid phase separation has diverse biological functions.•This review synthesizes how the lncRNAs may impact liquid–liquid phase separation.•Providing insights into potential applications leveraging liquid–liquid phase separation in the clinic.
The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently ...folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid–liquid phase separation of the entire protein.
TDP-43 is an RNA/DNA-binding protein which is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease. TDP-43's cytoplasmic mis-localization, liquid-liquid phase separation ...(LLPS) due to RNA depletion and aggregation, are proposedly important TDP-43-toxicity causing mechanisms. So far, therapeutic options for ALS are extremely ineffective hence, multi-faceted approaches such as targeting the oxidative stress and inhibiting the TDP-43's aggregation, are being actively pursued. Recently, we have identified an acridine derivative, AIM4, as an anti-TDP-43 aggregation molecule however, its mechanism is not deciphered. Here, we have utilized computational tools to examine binding site(s) of AIM4 in the TDP-43 structure and compared with other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic region (aa: 288–319), with Gly-288 & Phe-289 residues which are also important for TDP-43's LLPS. Importantly, alike to previously reported effects of RNA, AIM4 could also inhibit the in vitro LLPS of a C-terminal fragment TDP-432C bearing an A315T familial mutation. Furthermore, isothermal titration calorimetry (ITC) data also support the binding of AIM4 to TDP-432C-A315T. This antagonism of AIM4 towards TDP-43's LLPS and presence of binding site of AIM4 on TDP-43 support AIM4's potential to be an important molecule towards ALS therapeutic research.
Liquid-liquid phase separation (LLPS) in biology describes a process by which proteins form membraneless condensates within a cellular compartment when conditions are met, including the concentration ...and posttranslational modifications of the protein components, the condition of the aqueous solution (pH, ionic strength, pressure, and temperature), and the existence of assisting factors (such as RNAs or other proteins). In these supramolecular liquid droplet-like inclusion bodies, molecules are held together through weak intermolecular and/or intramolecular interactions. With the aid of LLPS, cells can assemble functional sub-units within a given cellular compartment by enriching or excluding specific factors, modulating cellular function, and rapidly responding to environmental or physiological cues. Hence, LLPS is emerging as an important means to regulate biology and physiology. Yet, excessive inclusion body formation by, for instance, higher-than-normal concentrations or mutant forms of the protein components could result in the conversion from dynamic liquid condensates into more rigid gel- or solid-like aggregates, leading to the disruption of the organelle's function followed by the development of human disorders like neurodegenerative diseases. In summary, well-controlled formation and de-formation of LLPS is critical for normal biology and physiology from single cells to individual organisms, whereas abnormal LLPS is involved in the pathophysiology of human diseases. In turn, targeting these aggregates or their formation represents a promising approach in treating diseases driven by abnormal LLPS including those neurodegenerative diseases that lack effective therapies.
Subcellular compartmentalization contributes to the organization of a plethora of molecular events occurring within cells. This can be achieved in membraneless organelles generated through ...liquid–liquid phase separation (LLPS), a demixing process that separates and concentrates cellular reactions. RNA is often a critical factor in mediating LLPS. Recent evidence indicates that DNA damage response foci are membraneless structures formed via LLPS and modulated by noncoding transcripts synthesized at DNA damage sites. Neurodegeneration is often associated with DNA damage, and dysfunctional LLPS events can lead to the formation of toxic aggregates. In this review, we discuss those gene products involved in neurodegeneration that undergo LLPS and their involvement in the DNA damage response.
Intracellular compartments can assemble as membraneless organelles through a demixing process known as ‘liquid–liquid phase separation’ (LLPS).DNA damage response foci are membraneless structures fueled by LLPS of some DNA damage response factors and are modulated by noncoding transcripts synthesized at DNA damage sites.Several forms of neurodegeneration are associated with, and possibly caused by, dysfunctional LLPS events, ultimately leading to the accumulation of toxic solid-like structures.Emerging evidence links factors involved in LLPS events and neurodegeneration with the cellular response to DNA damage.
TDP-43 proteinopathy is implicated in the neurodegenerative diseases, ALS and FTLD-TDP. Metal ion dyshomeostasis is observed in neurodegenerative diseases including ALS. Previously, mice expressing ...A315T familial ALS TDP-43 mutant showed elevated spinal cord Zn2+ levels. Recently, Zn2+ was observed to modulate the in vitro amyloid-like aggregation of the TDP-43's RRM12 domains. As a systematic knowledge of the TDP-43's interaction with Zn2+ is lacking, we in silico predicted potential Zn2+ binding sites in TDP-43 and estimated their relative solvent accessibilities. Zn2+ binding sites were predicted in the TDP-43's N-terminal domain, in the linker region between RRM1 and RRM2 domain, within RRM2 domain and at the junction of the RRM2 and C-terminal domain (CTD), but none in the 311–360 region of CTD. Furthermore, we found that Zn2+ promotes the in vitro thioflavin-T-positive aggregations of C-terminal fragments (CTFs) termed TDP-432C and TDP-432C-A315T that encompass the RRM2 and CTD domains. Also, while the Alexa-fluor fluorescently labelled TDP-432C and TDP-432C-A315T proteins manifested liquid-like spherical droplets, Zn2+ caused a solid-like phase separation that was not ameliorated even by carboxymethylation of the free cysteines thereby implicating the other Zn2+-binding residues. The observed Zn2+-promoted TDP-43 CTF's solid-like phase separation can be relevant to the Zn2+ dyshomeostasis in ALS and FTLD-TDP.
•Metal ion dyshomeostasis is a pathological feature of the TDP-43 pathology in ALS and FTLD-TDP.•Several Zn2+ binding sites are predicted here in the known domain structures of TDP-43 .•Zn2+ accelerated the in vitro aggregations of C-terminal fragments, TDP-432C and TDP-432C-A315T.•Zn2+ changes the in vitro LLPS of TDP-432C and TDP-432C-A315T into solid-like phase separation.•Free cysteines in TDP-432C and TDP-432C-A315T are not essential for the phase changing effects of Zn2+.
PDF
