Mutations in human prion-like domains: pathogenic but not always amyloidogenic.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are multifunctional proteins with integral roles in RNA metabolism and the regulation of alternative splicing. These proteins typically contain prion-like domains of low complexity (PrLDs or LCDs) that govern their assembly into either functional or pathological amyloid fibrils. To date, over 60 mutations targeting the LCDs of hnRNPs have been identified and associated with a spectrum of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD). The cryo-EM structures of pathological and functional fibrils formed by different hnRNPs have been recently elucidated, including those of hnRNPA1, hnRNPA2, hnRNPDL-2, TDP-43, and FUS. In this review, we discuss the structural features of these amyloid assemblies, placing particular emphasis on scrutinizing the impact of prevalent disease-associated mutations mapping within their LCDs. By performing systematic energy calculations, we reveal a prevailing trend of destabilizing effects induced by these mutations in the amyloid structure, challenging the traditionally assumed correlation between pathogenicity and amyloidogenic propensity. Understanding the molecular basis of this discrepancy might provide insights for developing targeted therapeutic strategies to combat hnRNP-associated diseases.

RNA and the RNA-binding protein FUS act in concert to prevent TDP-43 spatial segregation.

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.

Cryo-EM structures of the D290V mutant of the hnRNPA2 low-complexity domain suggests how D290V affects phase separation and aggregation.

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain. We find that the mutant form of hnRNPA2 differs from the WT fibrils in four ways. In contrast to the WT fibrils, the PY-nuclear localization signals in the fibril cores of all three mutant polymorphs are less accessible to chaperones. Also, the mutant fibrils are more stable than WT fibrils as judged by phase separation, thermal stability, and energetic calculations. Similar to other pathogenic amyloids, the mutant fibrils are polymorphic. Thus, these structures offer evidence to explain how a D-to-V missense mutation diverts the assembly of reversible, functional amyloid-like fibrils into the assembly of pathogenic amyloid, and may shed light on analogous conversions occurring in other ribonucleoproteins that lead to neurological diseases such as amyotrophic lateral sclerosis and frontotemporal dementia.

[Biological phase separation in neuromuscular diseases].

Biological phase separation refers to the liquid-liquid phase separation of biomolecules such as proteins in cells. Phase separation is driven by low-complexity domains of phase-separating proteins and strictly controlled by regulatory factors. Phase separation has also been found to be disrupted by genetic abnormalities. Abnormal aggregates of causative proteins accumulate in many neuromuscular diseases. In recent years, it has become clear that phase separating proteins are associated with neuromuscular diseases, and that abnormalities in the regulation of phase separation leads to the formation of aggregates. Gains in our knowledge of biological phase separation is gradually elucidating the pathogenesis of neuromuscular diseases.

How do disordered head domains assist in the assembly of intermediate filaments?

The dominant structural feature of intermediate filament (IF) proteins is a centrally located α-helix. These long α-helical segments become paired in a parallel orientation to form coiled-coil dimers. Pairs of dimers further coalesce in an anti-parallel orientation to form tetramers. These early stages of intermediate filament assembly can be accomplished solely by the central α-helices. By contrast, the assembly of tetramers into mature intermediate filaments is reliant upon an N-terminal head domain. IF head domains measure roughly 100 amino acids in length and have long been understood to exist in a state of structural disorder. Here, we describe experiments favoring the unexpected idea that head domains self-associate to form transient structural order in the form of labile cross-ß interactions. We propose that this weak form of protein structure allows for dynamic regulation of IF assembly and disassembly. We further offer that what we have learned from studies of IF head domains may represent a simple, unifying template for understanding how thousands of other intrinsically disordered proteins help to establish dynamic morphological order within eukaryotic cells.

"Boundary residues" between the folded RNA recognition motif and disordered RGG domains are critical for FUS-RNA binding.

Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the "boundary residues" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA.

PARIS undergoes liquid-liquid phase separation and poly(ADP-ribose)-mediated solidification.

ZNF746 was identified as parkin-interacting substrate (PARIS). Investigating its pathophysiological properties, we find that PARIS undergoes liquid-liquid phase separation (LLPS) and amorphous solid formation. The N-terminal low complexity domain 1 (LCD1) of PARIS is required for LLPS, whereas the C-terminal prion-like domain (PrLD) drives the transition from liquid to solid phase. In addition, we observe that poly(ADP-ribose) (PAR) strongly binds to the C-terminus of PARIS near the PrLD, accelerating its LLPS and solidification. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PAR formation leads to PARIS oligomerization in human iPSC-derived dopaminergic neurons that is prevented by the PARP inhibitor, ABT-888. Furthermore, SDS-resistant PARIS species are observed in the substantia nigra (SN) of aged mice overexpressing wild-type PARIS, but not with a PAR binding-deficient PARIS mutant. PARIS solidification is also found in the SN of mice injected with preformed fibrils of α-synuclein (α-syn PFF) and adult mice with a conditional knockout (KO) of parkin, but not if α-syn PFF is injected into mice deficient for PARP1. Herein, we demonstrate that PARIS undergoes LLPS and PAR-mediated solidification in models of Parkinson's disease.

Structural polymorphism of the low-complexity C-terminal domain of TDP-43 amyloid aggregates revealed by solid-state NMR.

Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43300-414), TDP-11 (TDP-43300-399), and TDP-10 (TDP-43314-414), adopt distinct polymorphic structures in their amyloid fibrillar state. Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.

Influence of ALS-linked M337V mutation on the conformational ensembles of TDP-43321-340 peptide monomer and dimer.

The transactive response (TAR) DNA/RNA-binding protein 43 (TDP-43) can self-assemble into both functional stress granules via liquid-liquid phase separation (LLPS) and pathogenic amyloid fibrillary aggregates that are closely linked to amyotrophic lateral sclerosis. Previous experimental studies reported that the low complexity domain (LCD) of TDP-43 plays an essential role in the LLPS and aggregation of the full-length protein, and it alone can also undergo LLPS to form liquid droplets mainly via intermolecular interactions in the 321-340 region. And the ALS-associated M337V mutation impairs LCD's LLPS and facilitates liquid-solid phase transition. However, the underlying atomistic mechanism is not well understood. Herein, as a first step to understand the M337V-caused LLPS disruption of TDP-43 LCD mediated by the 321-340 region and the fibrillization enhancement, we investigated the conformational properties of monomer/dimer of TDP-43321-340 peptide and its M337V mutant by performing extensive all-atom explicit-solvent replica exchange molecular dynamic simulations. Our simulations demonstrate that M337V mutation alters the residue regions with high helix/ß-structure propensities and thus affects the conformational ensembles of both monomer and dimer. M337V mutation inhibits helix formation in the N-terminal Ala-rich region and the C-terminal mutation site region, while facilitating their long ß-sheet formation, albeit with a minor impact on the average probability of both helix structure and ß-structure. Further analysis of dimer system shows that M337V mutation disrupts inter-molecular helix-helix interactions and W334-W334 π-π stacking interactions which were reported to be important for the LLPS of TDP-43 LCD, whereas enhances the overall peptide residue-residue interactions and weakens peptide-water interactions, which is conducive to peptide fibrillization. This study provides mechanistic insights into the M337V-mutation-induced impairment of phase separation and facilitation of fibril formation of TDP-43 LCD.

Evidence that only EWS among the FET proteins acquires a low partitioning property for the hyperosmotic stress response by O-GlcNAc glycosylation on its low-complexity domain.

FET proteins (FUS, EWS, and TAF15) share a common domain organization, bind RNA/DNA, and perform similarly multifunctional roles in the regulation of gene expression. Of the FET proteins, however, only EWS appears to have a distinct property in the cellular stress response. Therefore, we focused on the relationship between hyperosmotic stress response and post-translational modifications of the FET proteins. We confirmed that the hyperosmotic stress-dependent translocation from the nucleus to the cytoplasm and the cellular granule formation of FET proteins, and that EWS is less likely to partition into cellular granules in the cytoplasm than FUS or TAF15. The domain involved in the less partitioning property of EWS was found to be its low-complexity domain (LCD). Chemoenzymatic labeling analysis of O-linked ß-N-acetylglucosamine (O-GlcNAc) residues revealed that O-GlcNAc glycosylation occurs frequently in the LCD of EWS. A correlation was observed between the glycosylation of EWS and the less partitioning property under the hyperosmotic stress. These results suggest that among the FET proteins, only EWS has acquired the unique property through O-GlcNAc glycosylation. The glycosylation may play an essential role in regulating physiological functions of EWS, such as transcriptional activity, in addition to the property in cellular stress response.

Clinical and genetic characteristics of amyotrophic lateral sclerosis patients with ANXA11 variants.

Increasing genetic evidence supports the hypothesis that variants in the annexin A11 gene (ANXA11) contribute to amyotrophic lateral sclerosis pathogenesis. Therefore, we studied the clinical aspects of sporadic amyotrophic lateral sclerosis patients carrying ANXA11 variants. We also implemented functional experiments to verify the pathogenicity of the hotspot variants associated with amyotrophic lateral sclerosis-frontotemporal dementia. Korean patients diagnosed with amyotrophic lateral sclerosis (n = 882) underwent genetic evaluations through next-generation sequencing, which identified 16 ANXA11 variants in 26 patients. We analysed their clinical features, such as the age of onset, progression rate, initial symptoms and cognitive status. To evaluate the functional significance of the ANXA11 variants in amyotrophic lateral sclerosis-frontotemporal dementia pathology, we additionally utilized patient fibroblasts carrying frontotemporal dementia-linked ANXA11 variants (p.P36R and p.D40G) to perform a series of in vitro studies, including calcium imaging, stress granule dynamics and protein translation. The frequency of the pathogenic or likely pathogenic variants of ANXA11 was 0.3% and the frequency of variants classified as variants of unknown significance was 2.6%. The patients with variants in the low-complexity domain presented unique clinical features, including late-onset, a high prevalence of amyotrophic lateral sclerosis-frontotemporal dementia, a fast initial progression rate and a high tendency for bulbar-onset compared with patients carrying variants in the C-terminal repeated annexin homology domains. In addition, functional studies using amyotrophic lateral sclerosis-frontotemporal dementia patient fibroblasts revealed that the ANXA11 variants p.P36R and p.D40G impaired intracellular calcium homeostasis, stress granule disassembly and protein translation. This study suggests that the clinical manifestations of amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia spectrum patients with ANXA11 variants could be distinctively characterized depending upon the location of the variant.

Nab3 nuclear granule accumulation is driven by respiratory capacity.

Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules. Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery.

Expansion and functional analysis of the SR-related protein family across the domains of life.

Serine/arginine-rich (SR) proteins comprise a family of proteins that is predominantly found in eukaryotes and plays a prominent role in RNA splicing. A characteristic feature of SR proteins is the presence of an S/R-rich low-complexity domain (RS domain), often in conjunction with spatially distinct RNA recognition motifs (RRMs). To date, 52 human proteins have been classified as SR or SR-related proteins. Here, using an unbiased series of composition criteria together with enrichment for known RNA binding activity, we identified >100 putative SR-related proteins in the human proteome. This method recovers known SR and SR-related proteins with high sensitivity (∼94%), yet identifies a number of additional proteins with many of the hallmark features of true SR-related proteins. Newly identified SR-related proteins display slightly different amino acid compositions yet similar levels of post-translational modification, suggesting that these new SR-related candidates are regulated in vivo and functionally important. Furthermore, candidate SR-related proteins with known RNA-binding activity (but not currently recognized as SR-related proteins) are nevertheless strongly associated with a variety of functions related to mRNA splicing and nuclear speckles. Finally, we applied our SR search method to all available reference proteomes, and provide maps of RS domains and Pfam annotations for all putative SR-related proteins as a resource. Together, these results expand the set of SR-related proteins in humans, and identify the most common functions associated with SR-related proteins across all domains of life.

F/YGG-motif is an intrinsically disordered nucleic-acid binding motif.

Heterogeneous nuclear ribonucleoproteins (hnRNP) function in RNA processing, have RNA-recognition motifs (RRMs) and intrinsically disordered, low-complexity domains (LCDs). While RRMs are drivers of RNA binding, there is only limited knowledge about the RNA interaction by the LCD of some hnRNPs. Here, we show that the LCD of hnRNPA2 interacts with RNA via an embedded Tyr/Gly-rich region which is a disordered RNA-binding motif. RNA binding is maintained upon mutating tyrosine residues to phenylalanines, but abrogated by mutating to alanines, thus we term the RNA-binding region 'F/YGG motif'. The F/YGG motif can bind a broad range of structured (e.g. tRNA) and disordered (e.g. polyA) RNAs, but not rRNA. As the F/YGG otif can also interact with DNA, we consider it a general nucleic acid-binding motif. hnRNPA2 LCD can form dense droplets, by liquid-liquid phase separation (LLPS). Their formation is inhibited by RNA binding, which is mitigated by salt and 1,6-hexanediol, suggesting that both electrostatic and hydrophobic interactions feature in the F/YGG motif. The D290V mutant also binds RNA, which interferes with both LLPS and aggregation thereof. We found homologous regions in a broad range of RNA- and DNA-binding proteins in the human proteome, suggesting that the F/YGG motif is a general nucleic acid-interaction motif.

Bioinformatic identification of previously unrecognized amyloidogenic proteins.

Low-complexity domains (LCDs) of proteins have been shown to self-associate, and pathogenic mutations within these domains often drive the proteins into amyloid aggregation associated with disease. These domains may be especially susceptible to amyloidogenic mutations because they are commonly intrinsically disordered and function in self-association. The question therefore arises whether a search for pathogenic mutations in LCDs of the human proteome can lead to identification of other proteins associated with amyloid disease. Here, we take a computational approach to identify documented pathogenic mutations within LCDs that may favor amyloid formation. Using this approach, we identify numerous known amyloidogenic mutations, including several such mutations within proteins previously unidentified as amyloidogenic. Among the latter group, we focus on two mutations within the TRK-fused gene protein (TFG), known to play roles in protein secretion and innate immunity, which are associated with two different peripheral neuropathies. We show that both mutations increase the propensity of TFG to form amyloid fibrils. We therefore conclude that TFG is a novel amyloid protein and propose that the diseases associated with its mutant forms may be amyloidoses.

Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis.

Reorganization of cellular proteins into subcellular compartments, such as the concentration of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. One example of this is the induction of the yeast Nab3 transcription termination factor to rearrange from its pan-nucleoplasmic distribution to a granule at the nuclear periphery in response to nutrient limitation. Recent work in many cell types has shown that protein condensation in the nucleus is functionally important for transcription initiation, RNA processing, and termination. However, little is known about how subnuclear compartments form. Here, we have quantitatively analyzed this dynamic process in living yeast using a high-throughput computational tool and fluorescence microscopy. This analysis revealed that Nab3 granule accumulation varies in penetrance across yeast strains. A concentrated single granule is formed from at least a quarter of the nuclear Nab3 drawn from the rest of the nucleus. Levels of granule accumulation were inversely correlated with a growth defect in the absence of glucose. Importantly, the basis for some of the variation in penetrance was attributable to a defect in mitochondrial function. This publicly available computational tool provides a rigorous, reproducible, and unbiased examination of Nab3 granule accumulation that should be widely applicable to a variety of fluorescent images. Thousands of live cells can be readily examined enabling rigorous statistical verification of significance. With it, we describe a new feature of inducible subnuclear compartment formation for RNA-binding transcription factors and an important determinant of granule biogenesis.

Liquid-liquid phase separation of RBGD2/4 is required for heat stress resistance in Arabidopsis.

As sessile organisms, plants are highly sensitive to environmental stresses. In response to stresses, globally repressed translation initiation leads to stress granule (SG) formation. Protein liquid-liquid phase separation (LLPS) contributes to SG formation, but a direct link between protein LLPS and stress resistance has not yet been found in plants. Here, we report that two RNA-binding proteins, RBGD2 and RBGD4, function redundantly to improve heat resistance in Arabidopsis. RBGD2 and RBGD4 undergo LLPS in vitro and condense into heat-induced SGs in vivo via tyrosine residue array (TRA). Importantly, disrupting LLPS by mutating TRA abolishes RBGD2/4 condensation in SGs and impairs their protective function against heat stress (HS). Further study found that upon HS, the RBGD2/4 interaction network expands with additional SG proteins and heat-responsive mRNA. Our work shows a mechanistic basis that underlies protein LLPS in HS response in plants and suggests manipulation of protein LLPS as a general strategy to improve plant stress resistance.

Distinct roles of hnRNPH1 low-complexity domains in splicing and transcription.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.

The low-complexity domains of the KMT2D protein regulate histone monomethylation transcription to facilitate pancreatic cancer progression.

BACKGROUND: Liquid-liquid phase separation (LLPS) within the nucleus is directly linked to driving gene expression through transcriptional complexes. Histone lysine methyltransferase 2D (KMT2D) is widely present in many cancers. It is known to epigenetically stimulate the expression of genes associated with tumorigenesis and metastasis. Our analyses show that KMT2D possesses two distinct low-complexity domains (LCDs) capable of driving the assembly of membrane-less condensates. The dependence of the mechanisms underlying monomethylation of H3K4 on the LLPS microenvironment derived from KMT2D LCDs is unclear in tumor. METHODS: KMT2D LCD-depletion cells were used to investigate tumor cell proliferation, apoptosis, and migration. We identified some core proteins, including WDR5, RBBP5, and ASH2L, which are involved in the KMT2D-associated catalytic complex in KMT2D LCD-deficient cells to further elucidate the mechanism that decreases monomethylation of H3K4. We also evaluated the viability of KMT2D LCD-deficient cells in vivo. Finally, using 1,6-hexanediol (HD), an inhibitor of LLPS, we determined cell activities associated with KMT2D function in wild-type PANC-1 cells. RESULTS: Without the LLPS microenvironment in KMT2D LCD-deficient cells or wild-type PANC-1 cells treated with HD, the WDR5 protein was significantly less stable and the protein-protein interactions between the components of the KMT2D-enzyme complex were attenuated, impairing the formation of the complex. Moreover, with the decrease in H3K4me1 level at enhancers, transcription factors such as LIFR and KLF4 were markedly downregulated, effectively inhibiting tumor progression. In xenograft tumor models, PANC-1 cells lacking the KMT2D LCDs showed effectively suppressed tumor growth compared to normal cells. CONCLUSIONS: Our data indicate that the two low-complexity domains of the KMT2D protein could form a stable LLPS microenvironment, promoting the KMT2D catalysis of H3K4 monomethylation through stabilization of the WDR5 protein and KMT2D-enzyme complex. Therefore, finding ways to regulate the LLPS microenvironment will be benefitial for new cancer treatment strategies.

Entropy and Fractal Dimension Study of the TDP-43 Protein Low Complexity Domain Sequence in ALS Disease Severity and SARS-CoV-2 Gene Sequences in Virulence Variability.

The low complexity domain (LCD) sequence has been defined in terms of entropy using a 12 amino acid sliding window along a protein sequence in the study of disease-related genes. The amyotrophic lateral sclerosis (ALS)-related TDP-43 protein sequence with intra-LCD structural information based on cryo-EM data was published recently. An application of entropy and Higuchi fractal dimension calculations was described using the Znf521 and HAR1 sequences. A computational analysis of the intra-LCD sequence entropy and Higuchi fractal dimension values at the amino acid level and at the ATCG nucleotide level were conducted without the sliding window requirement. The computational results were consistent in predicting the intermediate entropy/fractal dimension value produced when two subsequences at two different entropy/fractal dimension values were combined. The computational method without the application of a sliding-window was extended to an analysis of the recently reported virulent genes-Orf6, Nsp6, and Orf7a-in SARS-CoV-2. The relationship between the virulence functionality and entropy values was found to have correlation coefficients between 0.84 and 0.99, using a 5% uncertainty on the cell viability data. The analysis found that the most virulent Orf6 gene sequence had the lowest nucleotide entropy and the highest protein fractal dimension, in line with extreme value theory. The Orf6 codon usage bias in relation to vaccine design was discussed.