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Functional bacterial amyloids play a crucial role in the formation of biofilms, which mediate chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC amyloid fibrillar protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone‐like protein FapA on this process. Using solution nuclear magnetic resonance (NMR), we recently showed that both FapC and FapA are intrinsically disordered proteins (IDPs). Here, the secondary structure propensities (SSPs) are compared to alphafold (DeepMind, protein structure prediction tool/algorithm: https://alphafold.ebi.ac.uk/) models. We further demonstrate that the FapA chaperone interacts with FapC and significantly slows down the formation of FapC fibrils. Our NMR titrations reveal ~ 18% of the resonances show FapA‐induced chemical shift perturbations (CSPs), which has not been previously observed, the largest being for A82, N201, C237, C240, A241, and G245. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the repeat‐1 (R1)/loop‐2 (L2) and L2/R3 transition areas and at the C‐terminus of FapC. Remarkably, ~ 90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC, the largest being for S63, A69, A80, and I92. A temperature‐dependent effect of FapA was observed on FapC by thioflavin T (ThT) and NMR experiments. This study provides a detailed understanding of the interaction between the FapA and FapC, shedding light on the regulation and slowing down of amyloid formation, and has important implications for the development of therapeutic strategies targeting biofilms and associated infections. [ABSTRACT FROM AUTHOR]

The genome of Arabidopsis thaliana encodes thousands of intrinsically disordered proteins (IDPs). Several IDPs, exemplified by H2B.8, LHP1, ADCP1, and EMB1579, regulate transcription and epigenetic silencing in various condensation-dependent manners. Diverse IDPs such as FCA, FLL2, FRI, and/or their associated long non-coding RNAs, regulate the transcription of FLC via phase separation. SE promotes Microprocessor activity via its condensation. The protein can be hijacked by SAID1 and 2 to form nuclear condensates, where SAID1/2 sequester primary miRNA precursors (pri-miRNAs) and inhibit Microprocessor activity. SAID1/2 promote phosphorylation of SE, which triggers its destruction by the 20S proteasome and reduces its binding affinity for HYL1, while isomerization of SE promotes its condensation and miRNA production. SGS3 constitutes a regulatory hub to control the production of 21–22 nt small interfering RNAs (siRNAs) via phase separation. SGS3 condensation is critical for the recruitment of RDR6 and RNA substrates. However, SGS3 association with its RNA substrates is enhanced by FVE, which also bridges SGS3/RDR6 and DRD4/DCL4 to facilitate siRNA production. Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) possess low sequence complexity of amino acids and display non-globular tertiary structures. They can act as scaffolds, form regulatory hubs, or trigger biomolecular condensation to control diverse aspects of biology. Emerging evidence has recently implicated critical roles of IDPs and IDR-contained proteins in nuclear transcription and cytoplasmic post-transcriptional processes, among other molecular functions. We here summarize the concepts and organizing principles of IDPs. We then illustrate recent progress in understanding the roles of key IDPs in machineries that regulate transcriptional and post-transcriptional gene silencing (PTGS) in plants, aiming at highlighting new modes of action of IDPs in controlling biological processes. Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) possess low sequence complexity of amino acids and display non-globular tertiary structures. They can act as scaffolds, form regulatory hubs, or trigger bimolecular condensation to control diverse aspects of biology. Emerging evidence has recently implicated critical roles of IDPs and IDR-contained proteins in nuclear transcription and cytoplasmic post-transcriptional processes, among other molecular functions. We here summarize the concepts and organizing principles of IDPs. We then illustrate recent progress in understanding the roles of key IDPs in machineries that regulate transcriptional and post-transcriptional gene silencing (PTGS) in plants, aiming at highlighting new modes of action of IDPs in controlling biological processes. [ABSTRACT FROM AUTHOR]

FapA is an accessory protein within the biofilm forming functional bacterial amyloid related fap-operon in Pseudomonas, and maybe a chaperone for FapC controlling its fibrillization. To allow further structural analysis, here we present a complete sequential assignment of 1Hamide, 13Cα, 13Cβ, and 15N NMR resonances for the functional form of the monomeric soluble FapA protein, comprising amino acids between 29 and 152. From these observed chemical shifts, the secondary structure propensities (SSPs) were determined. FapA predominantly adopts a random coil conformation, however, we also identified small propensities for α-helical and β-strand conformations. Notably, these observed SSPs are smaller compared to the ones we recently observed for the monomeric soluble FapC protein. These NMR results provide valuable insights into the activity of FapA in functional amyloid formation and regulation, that will also aid developing strategies targeting amyloid formation within biofilms and addressing chronic infections. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHsps) play diverse roles in the stress response and maintenance of cellular functions. The Ustilago maydis genome codes for few sHsps. Among these, Hsp12 has previously been demonstrated to be involved in the pathogenesis of the fungus by our group. In the present study we further investigated the biological function of the protein in the pathogenic development of U. maydis. Analysis of the primary amino acid sequence of Hsp12 in combination with spectroscopic methods to analyse secondary protein structures revealed an intrinsically disordered nature of the protein. We also carried out detailed analysis on the protein aggregation prevention activity associated with Hsp12. Our data suggest Hsp12 has trehalose‐dependent protein aggregation prevention activity. Through assaying the interaction of Hsp12 with lipid membranes in vitro we also showed the ability of U. maydis Hsp12 to induce stability in lipid vesicles. U. maydishsp12 deletion mutants exhibited defects in the endocytosis process and delayed completion of the pathogenic life cycle. Therefore, U. maydis Hsp12 contributes to the pathogenic development of the fungus through its ability to relieve proteotoxic stress during infection as well as its membrane‐stabilizing function. [ABSTRACT FROM AUTHOR]

Abstract A rapidly evolving understanding of phase separation in the biological and physical sciences has led to the redefining of virus-engineered replication compartments in many viruses with RNA genomes. Condensation of viral, host and genomic and subgenomic RNAs can take place to evade the innate immunity response and to help viral replication. Divergent viruses prompt liquid–liquid phase separation (LLPS) to invade the host cell. During HIV replication there are several steps involving LLPS. In this review, we characterize the ability of individual viral and host partners that assemble into biomolecular condensates (BMCs). Of note, bioinformatic analyses predict models of phase separation in line with several published observations. Importantly, viral BMCs contribute to function in key steps retroviral replication. For example, reverse transcription takes place within nuclear BMCs, called HIV-MLOs while during late replication steps, retroviral nucleocapsid acts as a driver or scaffold to recruit client viral components to aid the assembly of progeny virions. Overall, LLPS during viral infections represents a newly described biological event now appreciated in the virology field, that can also be considered as an alternative pharmacological target to current drug therapies especially when viruses become resistant to antiviral treatment.

Abstract Background Hepatitis E virus (HEV) is the cause of a liver disease hepatitis E. The translation product of HEV ORF2 has recently been demonstrated as a protein involved in multiple functions besides performing its major role of a viral capsid. As intrinsically disordered regions (IDRs) are linked to various essential roles in the virus’s life cycle, we analyzed the disorder pattern distribution of the retrieved ORF2 protein sequences by employing different online predictors. Our findings might provide some clues on the disorder-based functions of ORF2 protein that possibly help us in understanding its behavior other than as a HEV capsid protein. Results The modeled three dimensional (3D) structures of ORF2 showed the predominance of random coils or unstructured regions in addition to major secondary structure components (alpha helix and beta strand). After initial scrutinization, the predictors VLXT and VSL2 predicted ORF2 as a highly disordered protein while the predictors VL3 and DISOPRED3 predicted ORF2 as a moderately disordered protein, thus categorizing HEV-ORF2 into IDP (intrinsically disordered protein) or IDPR (intrinsically disordered protein region) respectively. Thus, our initial predicted disorderness in ORF2 protein 3D structures was in excellent agreement with their predicted disorder distribution patterns (evaluated through different predictors). The abundance of MoRFs (disorder-based protein binding sites) in ORF2 was observed that signified their interaction with binding partners which might further assist in viral infection. As IDPs/IDPRs are targets of regulation, we carried out the phosphorylation analysis to reveal the presence of post-translationally modified sites. Prevalence of several disordered-based phosphorylation sites further signified the involvement of ORF2 in diverse and significant biological processes. Furthermore, ORF2 structure-associated functions revealed its involvement in several crucial functions and biological processes like binding and catalytic activities. Conclusions The results predicted ORF2 as a protein with multiple functions besides its role as a capsid protein. Moreover, the occurrence of IDPR/IDP in ORF2 protein suggests that its disordered region might serve as novel drug targets via functioning as potential interacting domains. Our data collectively might provide significant implication in HEV vaccine search as disorderness in viral proteins is related to mechanisms involved in immune evasion.

Abstract Background A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. Results Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. Conclusions Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce “intractable” proteins, especially those containing large intrinsically disordered regions.

A rapidly evolving understanding of phase separation in the biological and physical sciences has led to the redefining of virus-engineered replication compartments in many viruses with RNA genomes. Condensation of viral, host and genomic and subgenomic RNAs can take place to evade the innate immunity response and to help viral replication. Divergent viruses prompt liquid–liquid phase separation (LLPS) to invade the host cell. During HIV replication there are several steps involving LLPS. In this review, we characterize the ability of individual viral and host partners that assemble into biomolecular condensates (BMCs). Of note, bioinformatic analyses predict models of phase separation in line with several published observations. Importantly, viral BMCs contribute to function in key steps retroviral replication. For example, reverse transcription takes place within nuclear BMCs, called HIV-MLOs while during late replication steps, retroviral nucleocapsid acts as a driver or scaffold to recruit client viral components to aid the assembly of progeny virions. Overall, LLPS during viral infections represents a newly described biological event now appreciated in the virology field, that can also be considered as an alternative pharmacological target to current drug therapies especially when viruses become resistant to antiviral treatment. [ABSTRACT FROM AUTHOR]

Background: Hepatitis E virus (HEV) is the cause of a liver disease hepatitis E. The translation product of HEV ORF2 has recently been demonstrated as a protein involved in multiple functions besides performing its major role of a viral capsid. As intrinsically disordered regions (IDRs) are linked to various essential roles in the virus's life cycle, we analyzed the disorder pattern distribution of the retrieved ORF2 protein sequences by employing different online predictors. Our findings might provide some clues on the disorder-based functions of ORF2 protein that possibly help us in understanding its behavior other than as a HEV capsid protein. Results: The modeled three dimensional (3D) structures of ORF2 showed the predominance of random coils or unstructured regions in addition to major secondary structure components (alpha helix and beta strand). After initial scrutinization, the predictors VLXT and VSL2 predicted ORF2 as a highly disordered protein while the predictors VL3 and DISOPRED3 predicted ORF2 as a moderately disordered protein, thus categorizing HEV-ORF2 into IDP (intrinsically disordered protein) or IDPR (intrinsically disordered protein region) respectively. Thus, our initial predicted disorderness in ORF2 protein 3D structures was in excellent agreement with their predicted disorder distribution patterns (evaluated through different predictors). The abundance of MoRFs (disorder-based protein binding sites) in ORF2 was observed that signified their interaction with binding partners which might further assist in viral infection. As IDPs/IDPRs are targets of regulation, we carried out the phosphorylation analysis to reveal the presence of post-translationally modified sites. Prevalence of several disordered-based phosphorylation sites further signified the involvement of ORF2 in diverse and significant biological processes. Furthermore, ORF2 structure-associated functions revealed its involvement in several crucial functions and biological processes like binding and catalytic activities. Conclusions: The results predicted ORF2 as a protein with multiple functions besides its role as a capsid protein. Moreover, the occurrence of IDPR/IDP in ORF2 protein suggests that its disordered region might serve as novel drug targets via functioning as potential interacting domains. Our data collectively might provide significant implication in HEV vaccine search as disorderness in viral proteins is related to mechanisms involved in immune evasion. [ABSTRACT FROM AUTHOR]

Background: The evolutionary rate of disordered protein regions varies greatly due to the lack of structural constraints. So far, few studies have investigated the presence/absence patterns of compositional bias, indicative of disorder, across phylogenies in conjunction with human disease. In this study, we report a genome-wide analysis of compositional bias association with disease in human proteins and their taxonomic distribution. Methods: The human genome protein set provided by the Ensembl database was annotated and analysed with respect to both disease associations and the detection of compositional bias. The Uniprot Reference Proteome dataset, containing 11297 proteomes was used as target dataset for the comparative genomics of a well-defined subset of the Human Genome, including 100 characteristic, compositionally biased proteins, some linked to disease. Results: Cross-evaluation of compositional bias and disease-association in the human genome reveals a significant bias towards biased regions in disease-associated genes, with charged, hydrophilic amino acids appearing as over-represented. The phylogenetic profiling of 17 disease-associated, proteins with compositional bias across 11297 proteomes captures characteristic taxonomic distribution patterns. Conclusions: This is the first time that a combined genome-wide analysis of compositional bias, disease-association and taxonomic distribution of human proteins is reported, covering structural, functional, and evolutionary properties. The reported framework can form the basis for large-scale, follow-up projects, encompassing the entire human genome and all known gene-disease associations.

Simple Summary: To understand complex cellular processes, the investigation of intrinsically disordered proteins that are involved in the most crucial mechanisms is of utmost importance. However, intrinsically disordered proteins lack a well-defined structure and exhibit vast flexibility. A prominent example is α-synuclein, that under pathological conditions, self-interacts and forms highly-ordered filamentous aggregates characteristic of neurodegenerative disorders, e.g., Parkinson's disease. Due to its broad conformational ensemble, its investigation is hampered, and suitable biophysical methods to elucidate underlying interactions and mechanisms on a molecular level are scarce. Investigating the aggregation process and prevailing kinetics is essential to gain insight into the causes of the pathological processes and eventually invent effective treatments. Here, we set out to investigate the accelerated aggregation process of α-synuclein and its disease mutants in the presence of ethanol. For the first time, we made use of rapid scan electron paramagnetic resonance spectroscopy to study the kinetics of α-synuclein aggregation. We were able to highlight differences between different protein variants and demonstrated that this approach outperforms conventional techniques in terms of sensitivity and rapidity of data acquisition and successfully demonstrated that this technique is suitable for studying intermolecular interactions with fast kinetics. Intrinsically disordered proteins (IDPs) are involved in most crucial cellular processes. However, they lack a well-defined fold hampering the investigation of their structural ensemble and interactions. Suitable biophysical methods able to manage their inherent flexibility and broad conformational ensemble are scarce. Here, we used rapid scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study the intermolecular interactions of the IDP α-synuclein (aS). aS aggregation and fibril deposition is the hallmark of Parkinson's disease, and specific point mutations, among them A30P and A53T, were linked to the early onset of the disease. To understand the pathological processes, research intensively investigates aS aggregation kinetics, which was reported to be accelerated in the presence of ethanol. Conventional techniques fail to capture these fast processes due to their limited time resolution and, thus, lose kinetic information. We have demonstrated that RS EPR spectroscopy is suitable for studying aS aggregation by resolving underlying kinetics and highlighting differences in fibrillization behavior. RS EPR spectroscopy outperforms traditional EPR methods in terms of sensitivity by a factor of 5 in our case while significantly reducing data acquisition time. Thus, we were able to sample short time intervals capturing single events taking place during the aggregation process. Further studies will therefore be able to shed light on biological processes proceeding on fast time scales. [ABSTRACT FROM AUTHOR]

Background: The evolutionary rate of disordered proteins varies greatly due to the lack of structural constraints. So far, few studies have investigated the presence/absence patterns of intrinsically disordered regions (IDRs) across phylogenies in conjunction with human disease. In this study, we report a genome-wide analysis of compositional bias association with disease in human proteins and their taxonomic distribution. Methods: The human genome protein set provided by the Ensembl database was annotated and analysed with respect to both disease associations and the detection of compositional bias. The Uniprot Reference Proteome dataset, containing 11297 proteomes was used as target dataset for the comparative genomics of a well-defined subset of the Human Genome, including 100 characteristic, compositionally biased proteins, some linked to disease. Results: Cross-evaluation of compositional bias and disease-association in the human genome reveals a significant bias towards low complexity regions in disease-associated genes, with charged, hydrophilic amino acids appearing as over-represented. The phylogenetic profiling of 17 disease-associated, low complexity proteins across 11297 proteomes captures characteristic taxonomic distribution patterns. Conclusions: This is the first time that a combined genome-wide analysis of low complexity, disease-association and taxonomic distribution of human proteins is reported, covering structural, functional, and evolutionary properties. The reported framework can form the basis for large-scale, follow-up projects, encompassing the entire human genome and all known gene-disease associations.

Abstract Background Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia. Results Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system. Conclusions Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization.

Background: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. Results: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. Conclusions: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce "intractable" proteins, especially those containing large intrinsically disordered regions. [ABSTRACT FROM AUTHOR]

Background: Specific subvariants of uveal melanoma (UM) are associated with increased rates of metastasis compared to other subvariants. BRCA1 (BReast CAncer gene 1)-associated protein-1 (BAP1) is encoded by a gene that has been linked to aggressive behavior in UM. Methods: We evaluated BAP1 for the presence of intrinsically disordered protein regions (IDPRs) and its protein–protein interactions (PPI). We evaluated specific sequence-based features of the BAP1 protein using a set of bioinformatic databases, predictors, and algorithms. Results: We show that BAP1's structure contains extensive IDPRs as it is highly enriched in proline residues (the most disordered amino acid; p-value < 0.05), the average percent of predicted disordered residues (PPDR) was 57.34%, and contains 9 disorder-based binding sites (ie. molecular recognition features (MoRFs)). BAP1's intrinsic disorder allows it to engage in a complex PPI network with at least 49 partners (p-value < 1.0 × 10−16). Conclusion: These findings show that BAP1 contains IDPRs and an intricate PPI network. Mutations in UM that are associated with the BAP1 gene may alter the function of the IDPRs embedded into its structure. These findings develop the understanding of UM and may provide a target for potential novel therapies to treat this aggressive neoplasm. [ABSTRACT FROM AUTHOR]

The dysbindin domain-containing protein 1 (DBNDD1) is a conserved protein among higher eukaryotes whose structure and function are poorly investigated so far. Here, we present the backbone and side chain nuclear magnetic resonance assignments for the human DBNDD1 protein. Our chemical-shift based secondary structure analysis reveals the human DBNDD1 as an intrinsically disordered protein. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) participate in many biological processes by interacting with other proteins, including the regulation of transcription, translation, and the cell cycle. With the increasing amount of disorder sequence data available, it is thus crucial to identify the IDP binding sites for functional annotation of these proteins. Over the decades, many computational approaches have been developed to predict protein-protein binding sites of IDP (IDP-PPIS) based on protein sequence information. Moreover, there are new IDP-PPIS predictors developed every year with the rapid development of artificial intelligence. It is thus necessary to provide an up-to-date overview of these methods in this field. In this paper, we collected 30 representative predictors published recently and summarized the databases, features and algorithms. We described the procedure how the features were generated based on public data and used for the prediction of IDP-PPIS, along with the methods to generate the feature representations. All the predictors were divided into three categories: scoring functions, machine learning-based prediction, and consensus approaches. For each category, we described the details of algorithms and their performances. Hopefully, our manuscript will not only provide a full picture of the status quo of IDP binding prediction, but also a guide for selecting different methods. More importantly, it will shed light on the inspirations for future development trends and principles.

The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs). [ABSTRACT FROM AUTHOR]

Background: Primary cilia are sensory organelles crucial for organ development. The pivotal structure of the primary cilia is a microtubule that is generated via tubulin polymerization reaction that occurs in the basal body. It remains to be elucidated how molecules with distinct physicochemical properties contribute to the formation of the primary cilia. Results: Here we show that brain expressed X-linked 1 (Bex1) plays an essential role in tubulin polymerization and primary cilia formation. The Bex1 protein shows the physicochemical property of being an intrinsically disordered protein (IDP). Bex1 shows cell density-dependent accumulation as a condensate either in nucleoli at a low cell density or at the apical cell surface at a high cell density. The apical Bex1 localizes to the basal body. Bex1 knockout mice present ciliopathy phenotypes and exhibit ciliary defects in the retina and striatum. Bex1 recombinant protein shows binding capacity to guanosine triphosphate (GTP) and forms the condensate that facilitates tubulin polymerization in the reconstituted system. Conclusions: Our data reveals that Bex1 plays an essential role for the primary cilia formation through providing the reaction field for the tubulin polymerization. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) are involved in most crucial cellular processes. However, they lack a well-defined fold hampering the investigation of their structural ensemble and interactions. Suitable biophysical methods able to manage their inherent flexibility and broad conformational ensemble are scarce. Here, we used rapid scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study the intermolecular interactions of the IDP α-synuclein (aS). aS aggregation and fibril deposition is the hallmark of Parkinson’s disease, and specific point mutations, among them A30P and A53T, were linked to the early onset of the disease. To understand the pathological processes, research intensively investigates aS aggregation kinetics, which was reported to be accelerated in the presence of ethanol. Conventional techniques fail to capture these fast processes due to their limited time resolution and, thus, lose kinetic information. We have demonstrated that RS EPR spectroscopy is suitable for studying aS aggregation by resolving underlying kinetics and highlighting differences in fibrillization behavior. RS EPR spectroscopy outperforms traditional EPR methods in terms of sensitivity by a factor of 5 in our case while significantly reducing data acquisition time. Thus, we were able to sample short time intervals capturing single events taking place during the aggregation process. Further studies will therefore be able to shed light on biological processes proceeding on fast time scales.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described. [ABSTRACT FROM AUTHOR]

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDβ) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies. • MBD-flanking disordered domains (NTD and ID) increase MBD structural stability and dsDNA binding affinity. • MBD-distant domains (TRD, CTDa and CTDb) hardly affect MBD structural stability and dsDNA binding properties. • MeCP2 contains four potential dsDNA binding sites; MBD and ID contain the main sites for dsDNA binding. • NTD-MBD-ID is the minimal construct for dsDNA binding studies, and full-length protein for transcription studies. • NTD-MBD-ID is appropriate for finding molecules recovering/inhibiting dsDNA interaction (Rett syndrome or MDS treatment). [ABSTRACT FROM AUTHOR]

Constantly increasing attention to bioengineered proteins has led to the rapid development of new functional targets. Here we present the biophysical and functional characteristics of the newly designed CaM/AMBN-Ct fusion protein. The two-domain artificial target consists of calmodulin (CaM) and ameloblastin C-terminus (AMBN-Ct). CaM as a well-characterized calcium ions (Ca2+) binding protein offers plenty of options in terms of Ca2+ detection in biomedicine and biotechnologies. Highly negatively charged AMBN-Ct belongs to intrinsically disordered proteins (IDPs). CaM/AMBN-Ct was designed to open new ways of communication synergies between the domains with potential functional improvement. The character and function of CaM/AMBN-Ct were explored by biophysical and molecular modelling methods. Experimental studies have revealed increased stability and preserved CaM/AMBN-Ct function. The results of molecular dynamic simulations (MDs) outlined different interface patterns between the domains with potential allosteric communication within the fusion. Unlabelled Image • The artificial CaM/AMBN-Ct fusion protein consists of calcium-binding CaM and an AMBN C-terminal IDP domain. • CaM in the CaM/AMBN-Ct fusion preserved its native folding and binding functions • The allosteric interplay of CaM and AMBN-Ct increased the thermostability of CaM • The CaM/AMBN-Ct fusion can potentially be used as a calcium sensor with increased thermal stability. [ABSTRACT FROM AUTHOR]

ABSTRACT A distinguishing morphological feature of all herpesviruses is the multiprotein tegument layer located between the nucleocapsid and lipid envelope of the virion. Tegument proteins play multiple roles in viral replication, including viral assembly, but we do not yet understand their individual functions or how the tegument is assembled and organized. UL11, the smallest tegument protein, is important for several distinct processes in replication, including efficient virion morphogenesis and cell-cell spread. However, the mechanistic understanding of its role in these and other processes is limited in part by the scant knowledge of its biochemical and structural properties. Here, we report that UL11 from herpes simplex virus 1 (HSV-1) is an intrinsically disordered, conformationally dynamic protein that undergoes liquid-liquid phase separation (LLPS) in vitro. Intrinsic disorder may underlie the ability of UL11 to exert multiple functions and bind multiple partners. Sequence analysis suggests that not only all UL11 homologs but also all HSV-1 tegument proteins contain intrinsically disordered regions of different lengths. The presence of intrinsic disorder, and potentially, the ability to form LLPS, may thus be a common feature of the tegument proteins. We hypothesize that tegument assembly may involve the formation of a biomolecular condensate, driven by the heterogeneous mixture of intrinsically disordered tegument proteins. IMPORTANCE Herpesvirus virions contain a unique tegument layer sandwiched between the capsid and lipid envelope and composed of multiple copies of about two dozen viral proteins. However, little is known about the structure of the tegument or how it is assembled. Here, we show that a conserved tegument protein UL11 from herpes simplex virus 1, a prototypical alphaherpesvirus, is an intrinsically disordered protein that undergoes liquid-liquid phase separation in vitro. Through sequence analysis, we find intrinsically disordered regions of different lengths in all HSV-1 tegument proteins. We hypothesize that intrinsic disorder is a common characteristic of tegument proteins and propose a new model of tegument as a biomolecular condensate.

The brain and acute leukemia cytoplasmic (BAALC; UniProt entry Q8WXS3) is a 180-residue-long human protein having six known isoforms. BAALC is expressed in either hematopoietic or neuroectodermal cells and its specific function is still to be revealed. However, as a presumably membrane-anchored protein at the cytoplasmic side it is speculated that BAALC exerts its function at the postsynaptic densities of certain neurons and might play a role in developing cytogenetically normal acute myeloid leukemia (CN-AML) when it is highly overexpressed by myeloid or lymphoid progenitor cells. In order to better understand the physiological role of BAALC and to provide the basis for a further molecular characterization of BAALC, we report here the 1H, 13C, and 15N resonance assignments for the backbone nuclei of its longest hematopoietic isoform (isoform 1). In addition, we present a 1HN and 15NH chemical shift comparison of BAALC with its shortest, neuroectodermal isoform (isoform 6) which shows only minor changes in the 1H and 15N chemical shifts. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) can engage in promiscuous interactions with their protein targets; however, it is not clear how this feature is encoded in the primary sequence of the IDPs and to what extent the surface properties and the shape of the binding cavity dictate the binding mode and the final bound conformation. Here we show, using a combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), that the promiscuous interaction of the intrinsically disordered regulatory domain of the mitogen-activated protein kinase kinase MKK4 with p38α and JNK1 is facilitated by folding-upon-binding into two different conformations, despite the high sequence conservation and structural homology between p38α and JNK1. Our results support a model whereby the specific surface properties of JNK1 and p38α dictate the bound conformation of MKK4 and that enthalpy–entropy compensation plays a major role in maintaining comparable binding affinities for MKK4 towards the two kinases.

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid–liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

Abstract As the group II LEA (late embryogenesis abundant) proteins, dehydrins (DHNs) play an important role in plant growth and development, as well as in response to abiotic or biotic stress challenges. In this study, a DHN gene named CsLEA11 was identified and characterized from Cucumis sativus. Sequence analysis of CsLEA11 showed that it is a Y3SK2-type DHN protein rich in hydrophilic amino acids. Expression analyses revealed that the transcription of CsLEA11 could be significantly induced by heat and cold stress. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-β-d-thiogalactoside (IPTG) was used to induce recombinant E. coli to express CsLEA11 gene. Overexpression of CsLEA11 in E. coli enhanced cell viability and conferred tolerance to heat and cold stress. Furthermore, CsLEA11 protein could protect the activity of lactate dehydrogenase (LDH) under heat stress. Taken together, our data demonstrate that CsLEA11 might function in tolerance of cucumber to heat and cold stress.

Recognition of the natural abundance and functional importance of intrinsically disordered proteins (IDPs), and protein hybrids that contain both intrinsically disordered protein regions (IDPRs) and ordered regions, is changing protein science. IDPs and IDPRs, i.e., functional proteins and protein regions without unique structures, can often be found in all organisms, and typically play vital roles in various biological processes. Disorder-based functionality complements the functions of ordered proteins and domains. However, by virtue of their existence, IDPs/IDPRs, which are characterized by remarkable conformational flexibility and structural plasticity, break multiple rules established over the years to explain structure, folding, and functionality of well-folded proteins with unique structures. Despite the general belief that unique biological functions of proteins require unique 3D-structures (which dominated protein science for more than a century), structure-less IDPs/IDPRs are functional, being able to engage in biological activities and perform impossible tricks that are highly unlikely for ordered proteins. With their exceptional spatio-temporal heterogeneity and high conformational flexibility, IDPs/IDPRs represent complex systems that act at the edge of chaos and are specifically tunable by various means. In this article, some of the wonders of intrinsic disorder are discussed as illustrations of their “mysterious” (meta)physics.

We report the changed levels of serum amyloid alpha, an immunologically active protein, in Parkinson’s disease (PD) patients’ peripheral tissues. We have previously shown that Saa-1 and -2 (serum amyloid alpha-1,-2, genes) were among the top downregulated genes in PD patients’ skin, using whole-genome RNA sequencing. In the current study, we characterized the gene and protein expression profiles of skin and blood samples from patients with confirmed PD diagnosis and age/sex matched controls. qRT-PCR analysis of PD skin demonstrated downregulation of Saa-1 and -2 genes in PD patients. However, the lowered amount of protein could not be visualized using immunohistochemistry, due to low quantity of SAA (Serum Amyloid Alpha, protein) in skin. Saa-1 and -2 expression levels in whole blood were below detection threshold based on RNA sequencing, however significantly lowered protein levels of SAA1/2 in PD patients’ serum were shown with ELISA, implying that SAA is secreted into the blood. These results show that SAA is differentially expressed in the peripheral tissues of PD patients.

The smallest open reading frame (ORF) encoded protein ORF3 of hepatitis E virus (HEV), recently, has been demonstrated to perform multiple functions besides accessory roles. ORF3 could act as a target for vaccine against HEV infections. The IDR (intrinsically disordered region); IDP (ID protein)/IDPR (ID protein region), plays critical role in various regulatory functions of viruses. The dark proteome of HEV-ORF3 protein including its structure and function was systematically examined by computer predictors to explicate its role in viral pathogenesis and drug resistance beyond its functions as accessory viral protein. Amino acid distribution showed ORF3 enrichment with disorder-promoting residues (Ala, Pro, Ser, Gly) while deficiency in order-promoting residues (Asn, Ile, Phe, Tyr and Trp). Initial investigation revealed ORF3 as IDP (entirely disordered protein) or IDPR (proteins consisting of IDRs with structured globular domains). Structural examination revealed preponderance of disordered regions interpreting ORF3 as moderately/highly disordered protein. Further disorder predictors categorized ORF3 as highly disordered protein/IDP. Identified sites and associated-crucial molecular functions revealed ORF3 involvement in diverse biological processes, substantiating them as targets of regulation. As ORF3 functions are yet to completely explored, thus, data on its disorderness could help in elucidating its disorder related functions., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© 2024 Biomedical Informatics.)

Prostate apoptosis response-4 (Par-4) is a proapoptotic tumor suppressor protein that has been linked to a large number of cancers. This 38 kilodalton (kDa) protein has been shown to be predominantly intrinsically disordered in vitro. In vivo, Par-4 is cleaved by caspase-3 at Asp-131 to generate the 25 kDa functionally active cleaved Par-4 protein (cl-Par-4) that inhibits NF-κB-mediated cell survival pathways and causes selective apoptosis in tumor cells. Here, we have employed circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) to assess the effects of various monovalent and divalent salts upon the conformation of cl-Par-4 in vitro. We have previously shown that high levels of sodium can induce the cl-Par-4 fragment to form highly compact, highly helical tetramers in vitro. Spectral characteristics suggest that most or at least much of the helical content in these tetramers are non-coiled coils. Here, we have shown that potassium produces a similar effect as was previously reported for sodium and that magnesium salts also produce a similar conformation effect, but at an approximately five times lower ionic concentration. We have also shown that anion identity has far less influence than does cation identity. The degree of helicity induced by each of these salts suggests that the “Selective for Apoptosis in Cancer cells” (SAC) domain—the region of Par-4 that is most indispensable for its apoptotic function—is likely to be helical in cl-Par-4 under the studied high salt conditions. Furthermore, we have shown that under medium-strength ionic conditions, a combination of high molecular weight aggregates and smaller particles form and that the smaller particles are also highly helical, resembling at least in secondary structure, the tetramers found at high salt.

Human T lymphotropic virus type 1 (HTLV-1) causes adult T cell leukemia and lymphoma and other neuroinflammatory diseases. The pX region of HTLV-1 genome encodes an accessory protein p30 that is required for viral persistence and spread in the host. p30 regulates viral gene expression at the transcription level by competing with Tax for p300 binding and at posttranscriptional level by nuclear retention of tax/rex messenger RNA (mRNA). In addition, p30 modulates the host cellular environment by binding to various host proteins such as ATM, REGγ, and PRMT5. However, the low expression levels of p30 has been a major hurdle in studying its structure-function relationship in the context of HTLV-1 pathobiology, which is most likely due to its intrinsically disordered nature. To investigate the unstable nature of p30, flow cytometric analysis of p30-GFP fusion protein expressed in Escherichia coli was conducted and bioinformatics analysis of p30 was performed. The bacterial cells were green fluorescent protein (GFP) positive, indicating that p30-GFP was in the soluble fraction. Induction, particularly at higher temperature, reduced the expression of p30-GFP. Moreover, p30-GFP was detected exclusively in insoluble fraction upon cell lysis, suggesting its unstable and disordered nature. The bioinformatics analysis of p30 protein sequence and amino acid content revealed that p30 has highly disordered regions from amino acids 75–155 and 197–241. Furthermore, p30 has regions for macromolecular interactions that could stabilize it and these regions coincide with the unstable regions. Collectively, the study indicates that HTLV-1 p30 is an intrinsically disordered protein. [ABSTRACT FROM AUTHOR]

Abstract The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that function in most stages of RNA metabolism. The prototypical member, hnRNP A1, is composed of three major domains; tandem N-terminal RNA Recognition Motifs (RRMs) and a C-terminal mostly intrinsically disordered region. HnRNP A1 is broadly implicated in basic cellular RNA processing events such as splicing, stability, nuclear export and translation. Due to its ubiquity and abundance, hnRNP A1 is also frequently usurped to control viral gene expression. Deregulation of the RNA metabolism functions of hnRNP A1 in neuronal cells contributes to several neurodegenerative disorders. Because of these roles in human pathologies, the study of hnRNP A1 provides opportunities for the development of novel therapeutics, with disruption of its RNA binding capabilities being the most promising target. The functional diversity of hnRNP A1 is reflected in the complex nature by which it interacts with various RNA targets. Indeed, hnRNP A1 binds both structured and unstructured RNAs with binding affinities that span several magnitudes. Available structures of hnRNP A1-RNA complexes also suggest a degree of plasticity in molecular recognition. Given the reinvigoration in hnRNP A1, the goal of this review is to use the available structural biochemical developments as a framework to interpret its wide-range of RNA functions. [ABSTRACT FROM AUTHOR]

We report the changed levels of serum amyloid alpha, an immunologically active protein, in Parkinson's disease (PD) patients' peripheral tissues. We have previously shown that Saa-1 and -2 (serum amyloid alpha-1,-2, genes) were among the top downregulated genes in PD patients' skin, using whole-genome RNA sequencing. In the current study, we characterized the gene and protein expression profiles of skin and blood samples from patients with confirmed PD diagnosis and age/sex matched controls. qRT-PCR analysis of PD skin demonstrated downregulation of Saa-1 and -2 genes in PD patients. However, the lowered amount of protein could not be visualized using immunohistochemistry, due to low quantity of SAA (Serum Amyloid Alpha, protein) in skin. Saa-1 and -2 expression levels in whole blood were below detection threshold based on RNA sequencing, however significantly lowered protein levels of SAA1/2 in PD patients' serum were shown with ELISA, implying that SAA is secreted into the blood. These results show that SAA is differentially expressed in the peripheral tissues of PD patients. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins and regions with their associated short linear motifs play key roles in transcriptional regulation. The disordered MYC-interaction motif (MIM) mediates interactions between MYC and MYB transcription factors in Arabidopsis thaliana that are critical for constitutive and induced glucosinolate (GLS) biosynthesis. GLSs comprise a class of plant defense compounds that evolved in the ancestor of the Brassicales order. We used a diverse set of search strategies to discover additional occurrences of the MIM in other proteins and in other organisms and evaluate the findings by means of structural predictions, interaction assays, and biophysical experiments. Our search revealed numerous MIM instances spread throughout the angiosperm lineage. Experiments verify that several of the newly discovered MIM-containing proteins interact with MYC TFs. Only hits found within the same transcription factor family and having similar characteristics could be validated, indicating that structural predictions and sequence similarity are good indicators of whether the presence of a MIM mediates interaction. The experimentally validated MIMs are found in organisms outside the Brassicales order, showing that MIM function is broader than regulating GLS biosynthesis.

Myelin basic protein (MBP) is an intrinsically disordered protein and in the central nervous system (CNS) mainly responsible for connecting the cytoplasmic surfaces of the multilamellar, compact myelin. Increased posttranslational modification of MBP is linked to both, the natural development (from adolescent to adult brains) of myelin, and features of multiple sclerosis. Here, we study how a combination of this intrinsically disordered myelin protein with varying the natural cholesterol content may alter the characteristics of myelin-like membranes and interactions between these membranes. Large unilamellar vesicles (LUVs) with a composition mimicking the cytoplasmic leaflet of myelin were chosen as the model system, in which different parameters contributing to the interactions between the lipid membrane and MBP were investigated. While we use cryo-transmission electron microscopy (TEM) for imaging, dynamic light scattering (DLS) and electrophoretic measurements through continuously-monitored phase-analysis light scattering (cmPALS) were used for a more global overview of particle size and charge, and electron paramagnetic resonance (EPR) spectroscopy was utilized for local behavior of lipids in the vesicles' membranes in aqueous solution. The cholesterol content was varied from 0 60 % in these LUVs and measurements were performed in the presence and absence of MBP. We find that the composition of the lipid layers is relevant to the interaction with MBP. Not only the size, the shape and the aggregation behavior of the vesicles depend on the cholesterol content, but also within each membrane, cholesterol's freedom of movement, its environmental polarity and its distribution were found to depend on the content using the EPR-active spin-labeled cholesterol (CSOSL). In addition, DLS and EPR measurements probing the transition temperatures of the lipid phases allow a correlation of specific behavior with the human body temperature of 37 °C. Overall, our results aid in understanding the importance of the native cholesterol content in the healthy myelin membrane, which serves as the basis for stable and optimum protein-bilayer interactions. Although studied in this specific myelin-like system, from a more general and materials science-oriented point of view, we could establish how membrane and vesicle properties depend on cholesterol and/or MBP content, which might be useful generally when specific membrane and vesicle characteristics are sought for. [Display omitted] • Cholesterol content impacts shape and aggregation behavior of LUVs interacting with MBP. • With MBP the rotational motion, the environmental polarity and the distribution of spin-labeled cholesterol change. • 44 % cholesterol content (natural content) in LUVs differs peculiarly as compared to 20 % or 60 % cholesterol content. [ABSTRACT FROM AUTHOR]

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context.

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impα3 and its depleted species, ∆Impα3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp α3 with a low micromolar affinity (~5 μM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target.

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs) represent approximately 30% of the human genome and play key roles in cell proliferation and cellular signaling by modulating the function of target proteins via protein–protein interactions. In addition, IDPs are involved in various human disorders, such as cancer, neurodegenerative diseases, and amyloidosis. To understand the underlying molecular mechanism of IDPs, it is important to study their structural features during their interactions with target proteins. However, conventional biochemical and biophysical methods for analyzing proteins, such as X-ray crystallography, have difficulty in characterizing the features of IDPs because they lack an ordered three-dimensional structure. Here, we present biochemical and biophysical studies on nucleolar phosphoprotein 140 (Nopp140), which mostly consists of disordered regions, during its interaction with casein kinase 2 (CK2), which plays a central role in cell growth. Surface plasmon resonance and electron paramagnetic resonance studies were performed to characterize the interaction between Nopp140 and CK2. A single-molecule fluorescence resonance energy transfer study revealed conformational change in Nopp140 during its interaction with CK2. These studies on Nopp140 can provide a good model system for understanding the molecular function of IDPs. [ABSTRACT FROM AUTHOR]

The control of supramolecular complexes in living systems at the molecular level is an important goal in life-sciences. Spatiotemporal organization of molecular distribution & flow of such complexes are essential physicochemical processes in living cells and important for pharmaceutical processes. Membraneless organelles (MO) found in eukaryotic cells, formed by liquid-liquid phase-separation (LLPS) of intrinsically disordered proteins (IDPs) control and adjust intracellular organization. Artificially designed compartments based on LLPS open up a novel pathway to control chemical flux and partition in vitro and in vivo. We designed a library of chemically precisely defined block copolymer-like proteins based on elastin-like proteins (ELPs) with defined charge distribution and type, as well as polar and hydrophobic block domains. This enables the programmability of physicochemical properties and to control adjustable LLPS in vivo attaining control over intracellular partitioning and flux as role model for in vitro and in vivo applications. Tailor-made ELP-like block copolymer proteins exhibiting IDP-behavior enable LLPS formation in vitro and in vivo allowing the assembly of membrane-based and membraneless superstructures via protein phase-separation in E. coli. Subsequently, we demonstrate the responsiveness of protein phase-separated spaces (PPSSs) to environmental physicochemical triggers and their selective, charge-dependent and switchable interaction with DNA or extrinsic and intrinsic molecules enabling their selective shuttling across semipermeable phase boundaries including (cell)membranes. This paves the road for adjustable artificial PPSS-based storage and reaction spaces and the specific transport across phase boundaries for applications in pharmacy and synthetic biology. • An amphiphilic protein (aELP) library allows for adjustable compartments in vivo. • Extrinsic stimuli modulate protein-phase separated spaces (PPSS) in E. coli. • PPSS formed by charged aELPs direct DNA localization. • Charged aELP forming PPSS can control molecular flux of molecules in vivo. • Responsive PPSS can be used in pharmaceutical applications and synthetic biology. [ABSTRACT FROM AUTHOR]

Phosphorus-31 (31P) NMR can be used to characterize the structure and dynamics of phosphorylated proteins. Here, I use 31P NMR to report on the chemical nature of a phosphothreonine that lies in the RNA binding domain of SLBP (stem-loop binding protein). SLBP is an intrinsically disordered protein and phosphorylation at this threonine promotes the assembly of the SLBP–RNA complex. The data show that the 31P chemical shift can be a good spectroscopic probe for phosphate-coupled folding and binding processes in intrinsically disordered proteins, particularly where the phosphate exhibits torsional strain and is involved in a network of hydrogen-bonding interactions.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

Prostate apoptosis response-4 (Par-4) is a 38 kDa largely intrinsically disordered tumor suppressor protein that functions in cancer cell apoptosis. Par-4 down-regulation is often observed in cancer while up-regulation is characteristic of neurodegenerative conditions such as Alzheimer’s disease. Cleavage of Par-4 by caspase-3 activates tumor suppression via formation of an approximately 25 kDa fragment (cl-Par-4) that enters the nucleus and inhibits Bcl-2 and NF-ƙB, which function in pro-survival pathways. Here, we have investigated the structure of cl-Par-4 using biophysical techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and intrinsic tyrosine fluorescence. The results demonstrate pH-dependent folding of cl-Par-4, with high disorder and aggregation at neutral pH, but a largely folded, non-aggregated conformation at acidic pH.

The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum. [ABSTRACT FROM AUTHOR]