Sedimentation Assays Research Articles

5H-benzo[c]fluorene derivative exhibits antiproliferative activity via microtubule destabilization

Present study aimed at a single component cyclization of 2-benzylidene-1-tetralones for the preparation of 5H-benzo[c]fluorenes and their antiproliferative activity. This ring closure reaction underwent via reductive cyclization in the presence of a sodium borohydride-aluminium chloride system. Ten diverse 5H-benzo[c]fluorene derivatives were prepared and evaluated for antiproliferative activity against three human cancer cell lines by SRB assay. Four of these benzofluorenes exhibited significant antiproliferative effect with an IC50 < 10.75 µM. The best representative compound 21, exhibited IC50 against K562 leukemic cells at 3.27 µM in SRB assay and 7.68 µM in Soft agar colony assay. It exhibited a microtubule destabilization effect in tubulin kinetics and inhibited 82.9 % microtubule polymer mass at 10 µM concentration in Protein Sedimentation assay (Microtubule). Compound 21 exerted G0/G1 phase arrest in cell division cycle analysis in K562 cells. It also induced apoptosis in K562 cells via activation of Caspase cascade pathway. Furthermore, compound 21 also possessed anti-inflammatory activity by inhibiting TNF-α and IL-6 moderately. It exhibited significant in vivo efficacy and reduced K562 tumour in xenograft mice by 47 % at an 80 mg/kg oral dose. Further, it was found to be safe and well tolerable up to 1000 mg/kg in Swiss albino mice. Compound 21 needs to be optimized for better in vivo efficacy in rodent models for further development.

Virulence factors associated with hyper virulent Klebsiella pneumoniae isolated from Different Clinical Sources

Klebsiella pneumonia is an opportunity pathogen causing several diseases including sepsis, pneumonia and wound infections. There are two pathotypes of Klebsiella pneumonia: classical K. pneumoniae (cKp) and hypervirulent K. pneumonia (hvkp). In this study, we attempted to discover the extent occurrence of hypervirulent K. pneumoniae strains among patients in Kirkuk city and their virulence factors. A total of 150 samples were collected from different hospitals in Kirkuk city (Kirkuk General Hospital, Azadi Teaching Hospital, Gynecological and Pediatric (Al-Nasr) Hospital and Children Hospital) during the period between November 2021 to June 2022. The age of patients ranged between 20- 60 years with both sexes. The results showed that 23.33% of K. pneumoniae isolates were positive for string test. Similar results were obtained by using sedimentation assay. The capsule of hvKp is a key virulence factor aiding in the bacterial dissemination and infection. For this reason, in current study, the capsular polysaccharide (CPS) was extracted and quantified. The results showed excessive production of capsular polysaccharide as seen in Klebsiella isolates 16,22,23,24,25,26,28 with an amount 49,41,50,74,49,49,73 μg/ml of CPS respectively. Siderophore production was also examined by using M9 minimal salts media. The results revealed that all K. pneumoniae isolates (100%) were able to produce siderophore. In addition, resistance to tellurite was also identified by using tellurite containing selective media and the results showed that 50% of isolates were resistant to tellurite and the remaining 50% were susceptible. A molecular study using conventional PCR revealed that 21 out of 30 isolates (70%) harbored the rmpA gene while the remaining 9 isolates (30%) lacked this gene. The presence of the iucA virulence gene was also detected and found to be present in all the isolates. Additionally, our findings indicated that 7 out of the 30 isolates (23%) were classified as hvkp.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate.

Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code.

Membrane protein chaperone and sodium chloride modulate the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aβ) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aβ aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aβ aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP-independent membrane protein chaperone that can both prevent and reverse Aβ aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aβ aggregation and determined that the chaperone prevents Aβ aggregation in a concentration-dependent manner. Size exclusion chromatography and sedimentation assays showed that 10-fold excess of cpSRP43 can keep Aβ in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aβ aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aβ aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aβ peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD.

Functional characterization of CpADF, an actin depolymerizing factor protein in Cryptosporidium parvum.

Cryptosporidium is a highly pathogenic water and food-borne zoonotic parasitic protozoan that causes severe diarrhea in humans and animals. Apicomplexan parasites invade host cells via a unique motility process called gliding, which relies on the parasite's microfilaments. Actin depolymerizing factor (ADF) is a fibrous-actin (F-actin) and globular actin (G-actin) binding protein essential for regulating the turnover of microfilaments. However, the role of ADF in Cryptosporidium parvum (C. parvum) remains unknown. In this study, we preliminarily characterized the biological functions of ADF in C. parvum (CpADF). The CpADF was a 135-aa protein encoded by cgd5_2800 gene containing an ADF-H domain. The expression of cgd5_2800 gene peaked at 12 h post-infection, and the CpADF was located in the cytoplasm of oocysts, middle region of sporozoites, and cytoplasm of merozoites. Neutralization efficiency of anti-CpADF serum was approximately 41.30%. Actin sedimentation assay revealed that CpADF depolymerized but did not undergo cosedimentation with F-actin and its ability of F-actin depolymerization was pH independent. These results provide a basis for further investigation of the roles of CpADF in the invasion of C. parvum.

Enhancement of capsular hypermucoviscosity in Klebsiella pneumoniae by Acanthamoeba.

Acanthamoeba and Klebsiella pneumoniae are both environmental commensals. Recently, clinical harm caused by hypermucoviscous K. pneumoniae has been observed. However, the interaction between these microbes and the origin of hypermucoviscous K. pneumoniae have not been reported. Here, we report that the bacterial capsule is enlarged when co-cultured with Acanthamoeba using India ink staining, and this effect depends on the number of parasites present. This interaction results in an enhancement of capsular polysaccharide production in the subsequent generations of K. pneumoniae, even without co-culturing with Acanthamoeba. The hypermucoviscosity of the capsule was examined using the sedimentation assay and string test. We also screened other K. pneumoniae serotypes, including K1, K2, K5, and K20, for interaction with Acanthamoeba using India ink staining, and found the same interaction effect. These findings suggest that the interaction between Acanthamoeba and K. pneumoniae could lead to harmful consequences in public health and nosocomial disease control, particularly hypermucoviscous K. pneumoniae infections.

Suppression of TDP-43 aggregation by artificial peptide binder targeting to its low complexity domain

TAR DNA-binding protein 43 (TDP-43), aggregation prone protein, is a potential target of drug discovery for amyotrophic lateral sclerosis. The molecular binders, targeting the disordered low complexity domain (LCD) relevant to the aggregation, may suppress the aggregation. Recently, Kamagata et al. developed a rational design of peptide binders targeting intrinsically disordered proteins based on contact energies between residue pairs. In this study, we designed 18 producible peptide binder candidates to TDP-43 LCD by using this method. Fluorescence anisotropy titration and surface plasmon resonance assays demonstrated that one of the designed peptides bound to TDP-43 LCD at 30 μM. Thioflavin-T fluorescence and sedimentation assays showed that the peptide binder suppressed the aggregation of TDP-43. In summary, this study highlights the potential applicability of peptide binder design for aggregation prone proteins.

Abstract 3563: SWI/SNF-associated DPF1 is a unique transcriptional regulator of malignant peripheral nerve sheath tumors

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are typically therapy resistant and associated with a poor prognosis. Genetic aberrations of the polycomb repressive complex 2 (PRC2) occur in up to 75% of MPNST cases. PRC2 functions antagonistically to SWI/SNF protein complexes, regulating expression of target loci in a dynamic relationship that is critical to normal cellular development and maintenance of cell identity. The consequences of PRC2 loss on chromatin state maintained by SWI/SNF in MPNST is unexplored. Therefore, this study used functional genomics to elucidate the role of SWI/SNF in MPNST. Methods: To define the transcriptional regulatory role of SWI/SNF complexes in MPNST, SWI/SNF components were targeted via a CRISPR knock-out (KO) screen combined with a single cell RNA sequencing (RNAseq) readout. Genes of interest highlighted by this screen were further investigated using bulk CRISPR RNAseq. Phenotypic effects of the expression of these genes were studied using loss-of-function assays coupled with colony formation assays. SWI/SNF complex heterogeneity was characterized using glycerol gradient sedimentation, co-immunoprecipitation (co-IP), and mass spectrometry experiments. Results: The investigation of SWI/SNF transcriptional regulation in MPNST highlighted the Double PHD Finger family proteins (DPF1,2,3) as regulating distinct downstream targets. Notably, DPF1 had a unique transcriptional profile compared to other SWI/SNF components. Bulk CRISPR KO RNAseq confirmed these findings and highlighted a specific set of DPF1 target genes including many long non-coding RNAs. The phenotypic role of DPF1 in MPNST was investigated using in vitro siRNA and CRISPR experiments, where DPF1 KO reduced proliferation and viability of MPNST cells in both 2D and 3D cell culture assays. Further, DPF1 was found contribute to anchorage independent cell growth of MPNST cells using soft agar assays. Glycerol gradient sedimentation assays demonstrated that DPF1 co-migrated with core components of a SWI/SNF complex known as canonical BAF (cBAF) and not the alternate GBAF and PBAF SWI/SNF complexes. These findings were further validated using co-immunoprecipitation assays where core the SWI/SNF ATPase, SMARCA4, pulled down DPF1, while components unique to the GBAF and PBAF complexes did not. An ATPase directed proteolysis targeting chimers (PROTAC) was used to therapeutically target cBAF in MPNST cells, reducing their growth and viability. Combination treatment with standard of care chemotherapy synergistically increased the efficacy of this drug. Conclusions: DPF1 was identified as a unique transcriptional regulator of MPNST cells and acts as a member of the cBAF SWI/SNF complex to play important phenotypic roles in this cancer type. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Jack F. Shern. SWI/SNF-associated DPF1 is a unique transcriptional regulator of malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3563.

Sustainable Microalgal Harvesting Process Applying Opuntia cochenillifera: Process Parameters Optimization

Microalgae harvesting by coagulation can use coagulant agents such as alum, synthetic polymers or biocoagulants. Biocoagulants have attracted the attention of researchers because they are natural, biodegradable, and promote high microalgal harvesting efficiencies. This study aims to optimize the harvesting of Chlorella vulgaris based on the dosage of the Opuntia cochenillifera extract and the choice of eluent for biopolymer extraction. The outdoor cultivation of C. vulgaris achieved a specific growth rate of 0.455 d−1 and a maximum biomass concentration of 1.28 gDW L−1. In order to harvest the microalgal biomass, the polymer present in the mucilage of O. cochenillifera was extracted using NaOH and HCl. Coagulation and sedimentation assays were performed with different coagulant dosages: 3.5, 5.9, and 8.2 g L−1. The maximum harvesting efficiencies using the acid and alkaline extract coagulant solutions were 80.8% and 99.5%, respectively, with a dosage of 3.5 g L−1. According to the results, the C. vulgaris biomass can be harvested with the mucilage from O. cochenillifera in acid and alkaline eluents. The application of this biocoagulant constitutes a sustainable solution for microalgal harvesting.

Inhibition of histone deacetylase 6 destabilizes ERK phosphorylation and suppresses cancer proliferation via modulation of the tubulin acetylation-GRP78 interaction

BackgroundThe leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown.MethodsThe clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT–PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells.ResultsHDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth.ConclusionsHDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Spherocytosis-Related L1340P Mutation in Ankyrin Affects Its Interactions with Spectrin.

Previously, we reported a new missense mutation in the ANK1 gene that correlated with the hereditary spherocytosis phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. Here, we report the molecular and physiological effects of this mutation. First, we assessed the binding activity of human β-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches-the study of association and dissociation responses of the spectrin-ankyrin binding domain and a sedimentation assay. In addition, we documented the changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of the L1340 aa residue for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the spectrin-binding activity of ankyrin.

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

Abstract B011: Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare and aggressive soft tissue sarcomas of the peripheral nervous system which are associated with a poor prognosis. Recurrent genetic mutations of the polycomb repressive complex 2 (PRC2) have been identified in up to 75% of MPNST cases. PRC2 is involved in an intricate epigenetic regulatory network in mammalian cells, where it is known to function antagonistically to the SWI/SNF protein complexes which are commonly associated with active transcription. The regulatory relationship between these complexes is critical to normal cell development and maintenance of cellular identity. However, little is known about how PRC2 aberrations affect this regulatory balance in MPNST. Therefore, this study aims to investigate the role of SWI/SNF in MPNST. Methods: A CRISPR knock-out (KO) screen combined with single cell RNA sequencing (RNAseq) was used to target 44 components of SWI/SNF complexes. The transcriptional regulatory effects of resulting genes of interest were further investigated via bulk CRISPR RNAseq experiments, while phenotypic effects were studied using loss-of-function assays coupled with colony formation assays. The dynamic complex formation was further deciphered using glycerol gradient sedimentation and co-immunoprecipitation (co-IP) assays. Results: Double PHD Fingers 1 gene, DPF1, was identified as a unique transcriptional regulator of MPNST via the single cell CRISPR KO screen. These findings were confirmed using a bulk RNAseq screen, where gene set enrichment analysis suggested DPF1 KO has an inhibitory effect on MPNST cell cycle and proliferation through enrichment of genes involved in the P53 pathway (FDR&amp;lt;0.0001) and the apoptotic pathway (FDR&amp;lt;0.0001). This hypothesis was validated using siRNA and CRISPR KO studies in MPNST cell lines, where DPF1 KO led to reduced proliferation and viability of these cells in both 2D and 3D cell culture assays. Soft agar assays were then used to highlight a role of DPF1 to in anchorage independent cell growth of MPNST cells. We then used glycerol gradient sedimentation assays to confirm DPF1 function as a member of the SWI/SNF complexes in MPNST, where DPF1 was observed to co-migrate with SMARCA4, a core ATPase commonly associated with the BAF complex of the SWI/SNF family. This assay was also used to eliminate DPF1 as a member of the GBAF and PBAF SWI/SNF complexes in MPNST, as we identified distinct sedimentation of DPF1 and proteins unique to these complexes. These findings were further validated using co-immunoprecipitation assays, where SMARCA4 had the ability to pull down DPF1, while components of the GBAF and PBAF complexes did not. Conclusions: DPF1 has been identified as a unique regulator of MPNST cells and acts as a member of the BAF SWI/SNF complex. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Michael C. Kelly, Jack F. Shern. Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B011.

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes, the Basis of Its Specificity and Bactericidal Activity.

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide–membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium2GN, Escherichia coli3GN, and Staphylococcus aureus3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.

Sedimentation Assays to Assess the Impact of Posttranslational Modifications on Phase Separation of RNA-Binding Proteins In Vitro and In Cells.

In the last years, RNA-binding proteins (RBPs) have been highlighted for their capacity to undergo liquid-liquid phase separation (LLPS). Aberrant phase transitions of RBPs from a liquid to a solid state are believed to underlie the formation of pathological RBP aggregates in several neurodegenerative diseases. Both in the physiological and the disease state, RBPs are often decorated with diverse posttranslational modifications (PTMs) that can influence the phase separation behavior, the physiological function, and the pathological behavior of the RBP. Here we describe two simple methods, sedimentation assays in vitro and in cells, that allow the analysis of RBP solubility as a measure of RBP phase separation in the absence or presence of a certain PTM.

ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis. <i>Spiroplasma eriocheiris</i>, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow P_i release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-P_i release states are unfavourable for them to remain as filaments.

GA signaling expands: The plant UBX domain-containing protein 1 is a binding partner for the GA receptor.

The plant Ubiquitin Regulatory X (UBX) domain-containing protein 1 (PUX1) functions as a negative regulator of gibberellin (GA) signaling. GAs are plant hormones that stimulate seed germination, the transition to flowering, and cell elongation and division. Loss of Arabidopsis (Arabidopsis thaliana) PUX1 resulted in a "GA-overdose" phenotype including early flowering, increased stem and root elongation, and partial resistance to the GA-biosynthesis inhibitor paclobutrazol during seed germination and root elongation. Furthermore, GA application failed to stimulate further stem elongation or flowering onset suggesting that elongation and flowering response to GA had reached its maximum. GA hormone partially repressed PUX1 protein accumulation, and PUX1 showed a GA-independent interaction with the GA receptor GA-INSENSITIVE DWARF-1 (GID1). This suggests that PUX1 is GA regulated and/or regulates elements of the GA signaling pathway. Consistent with PUX1 function as a negative regulator of GA signaling, the pux1 mutant caused increased GID1 expression and decreased accumulation of the DELLA REPRESSOR OF GA1-3, RGA. PUX1 is a negative regulator of the hexameric AAA+ ATPase CDC48, a protein that functions in diverse cellular processes including unfolding proteins in preparation for proteasomal degradation, cell division, and expansion. PUX1 binding to GID1 required the UBX domain, a binding motif necessary for CDC48 interaction. Moreover, PUX1 overexpression in cell culture not only stimulated the disassembly of CDC48 hexamer but also resulted in co-fractionation of GID1, PUX1, and CDC48 subunits in velocity sedimentation assays. Based on our results, we propose that PUX1 and CDC48 are additional factors that need to be incorporated into our understanding of GA signaling.

Dsk2 Induces Aggregation of Polyubiquitinated Proteins

The ubiquitin‐proteasome system (UPS) is an intricate system responsible for protein degradation in eukaryotic cells. Within the UPS, proteins targeted for degradation are tagged with polyubiquitin chains and subsequently recognized and degraded by the 26S proteasome. The three proteasome‐associated shuttle proteins Dsk2, Rad23, and Ddi1 mediate substrate degradation by helping deliver ubiquitin‐tagged substrates to the proteasome via a UBL domain that binds the proteasome and a UBA domain that binds to polyubiquitinated proteins. The importance of these shuttle proteins and the specific roles they play in proteasomal degradation are still being explored. To investigate the role of Dsk2 in proteasomal degradation, we examined degradation of model substrates by ∆Dsk2 proteasome in the presence or absence of purified Dsk2 protein. Surprisingly, we discovered that purified Dsk2 protein was able to remove ubiquitinated substrates from solution as determined using sedimentation assays. Dsk2’s ability to sediment substrates was dependent on ubiquitin chain linkage, with K63 linkages giving greater sedimentation than K48 or mixed linkages. Based on these findings, Dsk2‐induced substrate aggregation may play a role in the cellular fates of ubiquitinated proteins.

Three Novel Sequence Types Carbapenem-Resistant Klebsiella pneumoniae Strains ST5365, ST5587, ST5647 Isolated from Two Tertiary Teaching General Hospitals in Shanxi Province, in North China: Molecular Characteristics, Resistance and Virulence Factors.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) represents a significant threat to public health and has already drawn worldwide attention. Hence, we aim to comprehensively analyze the case condition, as well as molecular epidemiology, resistance and virulence of three CRKP isolates with new sequence types (STs).MethodsThree CRKP were collected from November 2019 to April 2021. The three patients’ clinical characteristics were analyzed through His system. In order to screen phenotype of metallo-carbapenemase, the modified Carbapenem Inactivation Method (mCIM) and EDTA-modified Carbapenem Inactivation Method (eCIM) were conducted. Three isolates were subjected to antimicrobial susceptibility testing (AST) using the agar dilution method or minimal broth dilution method. The string test, the sedimentation assay, biofilm formation and the serum resistance assay were performed as phenotypic experiments to assist in evaluating virulence. The presence of resistance and virulence genes were detected by Whole-Genome Sequencing (WGS). Serotypes and new STs were compared and determined by multi-locus sequence typing (MLST).ResultsOverall, all Klebsiella pneumoniae isolates were multi-resistant, but sensitive to tigecycline and colistin. Among them, all formed biofilms, strain 1 and strain 2 were classified as moderate-producers, while strain 3 as weak-producer. The results of the serum resistance assay indicated that only strain 2 was resistant. From WGS analysis, it showed that all isolates co-harbored multiple resistance genes, such as carbapenemase genes, sulfonamides, fluoroquinolones, aminoglycosides, and tetracyclines. Meanwhile, several virulence genes were also contained, including siderophores, fimbriae, capsule and lipopolysaccharides-associated genes. The serotypes of strain 1 and strain 2 manifested K35 and KL47, respectively.ConclusionThree novel ST5365, ST5587, ST5647 were first discovered in North China. Our study suggested that we should pay more attention to their resistance. And the results will help treat CRKP infections caused by these novel STs.