Enrichment of autophagic cargo by liquid–liquid phase separation allows the formation of distinct entities of macromolecular complexes to be degraded by selective autophagy.
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•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.
Coacervation is a liquid-liquid phase separation phenomenon. It involves the formation of a dense coacervate phase, rich in concentrated materials, and a co-existing immiscible dilute supernatant. ...This phenomenon can occur either from a homogeneous aqueous solution (simple coacervation) or when two different macromolecular aqueous solutions (proteins, polymers, and colloids) are brought into contact (complex coacervation). Coacervation has historical significance as it may have played a role in the origin of life, concentrating nutritious materials through liquid-liquid phase separation. It also reveals the underlying mechanisms of many biological phenomena such as intracellular biomolecular condensates, extracellular matrices, squid beak's gradient properties, sessile organism's wet adhesion, Alzheimer's diseases, and more. Coacervation provides insights and inspires promising solutions in areas like artificial cells/tissues, gradient materials, gene/drug delivery, underwater adhesives, and beyond. The driving forces of coacervation are noncovalent molecular interactions, often referred to as ‘chemistry beyond the molecule’, including hydrophobic interaction, electrostatic interaction, hydrogen-bonding interaction, cation-π interaction, π-π interaction, multivalency, etc. In this work, we have systematically reviewed the underlying noncovalent molecular interactions of simple coacervation and complex coacervation, respectively. We summarize commonly used materials and their corresponding molecular structures, discussing their applications. Some remaining challenge issues and perspectives for future studies are also presented. Understanding the underlying noncovalent molecular interactions of coacervation, alongside insights into molecular compositions and structures, can better guide the design of novel materials, elucidate various biological phenomena, and contribute to the development and optimization of relevant engineering technologies.
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NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation ...(LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.
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•dsRNA induces liquid-liquid phase separation (LLPS) of NLRP6 in vitro and in cells•ASC solidifies LLPS of NLRP6 and RNA to promote inflammasome activation•Poly-Lysine region in NLRP6 is critical for multivalent interaction and LLPS•Phase separation of NLRP6 promotes inflammasome activation in intestine and liver
dsRNA induces phase separation of NLRP6 for inflammasome activation.
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Co-amorphous drug delivery systems are evolving as a credible alternative to amorphous solid dispersions technology. In Co-amorphous systems (CAMs), a drug is stabilized in amorphous ...form using small molecular weight compounds called as co-formers. A wide variety of small molecular weight co-formers have been leveraged in the preparation of CAMs. The stability and supersaturation potential of prepared co-amorphous phases largely depend on the type of co-former employed in the CAMs. However, the rationality behind the co-former selection in co-amorphous systems is poorly understood and scarcely compiled in the literature. There are various facets to the rational selection of co-former for CAMs. In this context, the present review compiles various factors affecting the co-former selection. The factors have been broadly classified under Thermodynamic, Kinetic and Pharmacokinetic-Pharmacologically relevant parameters. In particular, the importance of Glass transition, Miscibility, Liquid-Liquid phase separation (LLPS), Crystallization inhibition has been deliberated in detail.
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•FASE is a catalogue of designer condensates for in vitro and in vivo use.•Scandinavian design principles help us to rethink/repurpose biomolecular condensation.•In vitro condensates ...have widespread use in industrial and medical applications.•In vivo condensates provide a platform for engineering novel biological function.
Protein phase separation has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomolecular condensates. While the implication of this physical phenomenon within cell biology is providing us with novel ways for understanding how cells compartmentalize biochemical reactions and encode function in such liquid-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biological matter. Here, we propose that understanding the cell’s instruction manual of phase separation will enable bioengineers to begin creating novel functionalized biological materials and unprecedented tools for synthetic biology. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the physical driving forces underlying phase separation, with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalogue and construction manual for the aspiring (membraneless) biomolecular architect. Our approach aims to inspire a new generation of bioengineers to rethink phase separation as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biological function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.
The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its ...considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3′-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.
TAR DNA binding protein 43 (TDP-43) is a versatile RNA/DNA binding protein involved in RNA-related metabolism. Hyper-phosphorylated and ubiquitinated TDP-43 deposits act as inclusion bodies in the ...brain and spinal cord of patients with the motor neuron diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While the majority of ALS cases (90-95%) are sporadic (sALS), among familial ALS cases 5-10% involve the inheritance of mutations in the
gene and the remaining (90-95%) are due to mutations in other genes such as:
, and
etc. Strikingly however, the majority of sporadic ALS patients (up to 97%) also contain the TDP-43 protein deposited in the neuronal inclusions, which suggests of its pivotal role in the ALS pathology. Thus, unraveling the molecular mechanisms of the TDP-43 pathology seems central to the ALS therapeutics, hence, we comprehensively review the current understanding of the TDP-43's pathology in ALS. We discuss the roles of TDP-43's mutations, its cytoplasmic mis-localization and aberrant post-translational modifications in ALS. Also, we evaluate TDP-43's amyloid-like
aggregation, its physiological vs. pathological oligomerization
, liquid-liquid phase separation (LLPS), and potential prion-like propagation propensity of the TDP-43 inclusions. Finally, we describe the various evolving TDP-43-induced toxicity mechanisms, such as the impairment of endocytosis and mitotoxicity etc. and also discuss the emerging strategies toward TDP-43 disaggregation and ALS therapeutics.
Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. ...Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.
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•Arginine-rich peptides undergo LLPS dependent on counterions or polyaromates•Toxic arginine-rich DPRs perturb stress granule dynamics and protein content•PR-induced stress granule formation is dependent on eIF2α phosphorylation and G3BP•PR promotes aggregation of ALS-related proteins containing prion-like domains
Proteins high in arginine content are enriched in stress granules. Boeynaems et al. show that arginine-rich peptides can actively mediate liquid-liquid phase separation, a process underlying liquid organelle formation. Toxic C9orf72 DPRs change the arginine content and alter the properties of stress granules.
The dual-specificity kinase DYRK3 controls the formation and dissolution of multiple biomolecular condensates, regulating processes including stress recovery and mitotic progression. Here, we report ...that DYRK3 functionally interacts with proteins associated with endoplasmic reticulum (ER) exit sites (ERESs) and that inhibition of DYRK3 perturbs the organization of the ERES-Golgi interface and secretory trafficking. DYRK3-mediated regulation of ERES depends on the N-terminal intrinsically disordered region (IDR) of the peripheral membrane protein SEC16A, which co-phase separates with ERES components to form liquid-like condensates on the surface of the ER. By modulating the liquid-like properties of ERES, we show that their physical state is essential for functional cargo trafficking through the early secretory pathway. Our findings support a mechanism whereby phosphorylation by DYRK3 and its reversal by serine-threonine phosphatases regulate the material properties of ERES to create a favorable physicochemical environment for directional membrane traffic in eukaryotic cells.
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•The kinase DYRK3 functionally interacts with ERES proteins•SEC16A phase separation drives formation of ERES•DYRK3 and phosphatase activity regulates the material state of ERES•The liquid-like state of ERES is required for secretory trafficking
Gallo and Rai et al. report that DYRK3 regulates the integrity and properties of ER exit sites (ERESs), membrane-associated protein assemblies mediating cargo export from the ER. They show that SEC16A phase separation drives ERES formation and that a liquid-like state of ERES, maintained by DYRK3, is necessary for function.
Interest in RNA damage as a novel threat associated with several human pathologies is rapidly increasing. Knowledge on damaged RNA recognition, repair, processing and decay is still scanty. ...Interestingly, in the last few years, more and more evidence put a bridge between DNA damage repair enzymes and the RNA world. The Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) was firstly identified as a crucial enzyme of the base excision repair (BER) pathway preserving genome stability toward non-distorting DNA lesion-induced damages. Later, an unsuspected role of APE1 in controlling gene expression was discovered and its pivotal involvement in several human pathologies, ranging from tumor progression to neurodegenerative diseases, has emerged. Recent novel findings indicate a role of APE1 in RNA metabolism, particularly in processing activities of damaged (abasic and oxidized) RNA and in the regulation of oncogenic microRNAs (miRNAs). Even though the role of miRNAs in human pathologies is well-known, the mechanisms underlying their quality control are still totally unexplored. A detailed knowledge of damaged RNA decay processes in human cells is crucial in order to understand the molecular processes involved in multiple pathologies. This cutting-edge perspective article will highlight these emerging aspects of damaged RNA processing and decay, focusing the attention on the involvement of APE1 in RNA world.
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