Granule-associated Proteins Research Articles

The evolution of granular surface structure and functional properties in rice starch during grain filling

The developmental changes in the granular surface structure and functional properties of starch during the entire grain filling period of rice (around 40 days) were investigated. The specific surface area of rice starch significantly decreased firstly then stabilized during growth due to increasing granular size. The pore volume decreased from 5.40 cm3/g at 6th day after anthesis (DAA-6) to 3.02 cm3/g (DAA-46). More starch granule-associated proteins (SGAPs) accumulated on the surface and in channels. Swelling power decreased by 46 %, whereas the flow behavior index (n) decreased by 32 % in upward curve during starch development from DAA-6 to DAA-30. Tan δ first dropped then remained steady at DAA 22–34 and lightly rebounded at the final stage, indicating that starch in the middle stage tended to have greater viscoelastic gel behavior at all sweeps. Mature starch showed lower in vitro hydrolysis rate and exhibited stronger enzymatic resistance. The results showed that granular surface features of rice starch may be an essential factor in determining rheological behavior and resistance to hydrolysis.

Diverse Phenotypic Presentation of the Welander Distal Myopathy Founder Mutation, With Myopathy and Amyotrophic Lateral Sclerosis in the Same Family.

Welander distal myopathy is a rare myopathy with prominent and early involvement of distal upper extremity muscles, prevalent in individuals of Scandinavian origin, and caused by a founder mutation in the cytotoxic granule-associated RNA-binding protein (T-cell intracellular antigen-1; TIA1), E384K. Different pathogenic variants in the TIA1 gene, distinct from the founder 1, have recently been associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), suggesting that TIA1-related disorders belong to the group of multisystem proteinopathies. We describe the first case of a two-generation family with the founder E384K TIA1 mutation demonstrating phenotypic variability; the mother manifested as Welander myopathy, whereas 2 daughters manifested as ALS. No other genetic cause of ALS was found in 1 of the affected daughters. We also discuss the possible mechanisms explaining this pleotropic presentation of the founder mutation.

Unraveling the impact of starch granule-associated proteins on the emulsifying ability of quinoa starch granules at multiple scales

Total starch granule-associated proteins (tGAP), including granule-channel (GCP) and granule-surface proteins (GSP), alter the physicochemical properties of starches. Quinoa starch (QS) acts as an effective emulsifier in Pickering emulsion. However, the correlation between the tGAP and the emulsifying capacity of QS at different scales remains unclear. Herein, GCP and tGAP were selectively removed from QS, namely QS-C and QS-A. Results indicated that the loss of tGAP increased the water permeability and hydrophilicity of the starch particles. Mesoscopically, removing tGAP decreased the diffusion rate and interfacial viscous modulus. Particularly, GSP had a more profound impact on the interfacial modulus than GCP. Microscopically and macroscopically, the loss of tGAP endowed QS with weakened emulsifying ability in terms of emulsions with larger droplet size and diminished rheological properties. Collectively, this work demonstrated that tGAP played an important role in the structural and interfacial properties of QS molecules and the stability of QS-stabilized emulsions.

Transglutaminase and protease: How starch granule-associated protein modification affects starch retrogradation

This study investigated the effects of transglutaminase (TG) and protease on starch granule-associated proteins and their impact on starch properties, especially retrogradation. TG incorporated storage protein onto the starch surface, while protease removed starch-granule associated proteins from it. Neither treatment altered the basic structural characteristics of starch such as its crystallinity or thermal properties, but could significantly affect swelling power, amylose leaching, and retrogradation. TG-treated wheat starch (WST) showed reduced swelling power (11.50) and amylose leaching (28.92 mg/g) compared with the control wheat starch (WSC) at 11.95 and 30.53 mg/g, respectively, suggesting a more stable structure. Conversely, protease-treated starch (WSP) exhibited increased swelling power at 12.47 and amylose leaching at 33.17 mg/g, suggesting a disintegration of starch granules. Both WSC and WST showed the same degree of retrogradation at the equilibrium state (a) at 0.06, whereas WSP, with a higher degree at 0.08, promoted retrogradation. The rate constant (k) for WST was lower (1.41 d−1) than that for WSC (2.37 d−1), indicating that TG treatment effectively inhibited retrogradation. These findings illustrated the distinct mechanisms through which TG and protease modify starch properties by acting on starch granule-associated proteins. This also highlighted the potential of TG and protease to control starch modifications for specific applications.

Effect of Mild Alkali Treatment on the Structure and Physicochemical Properties of Normal and Waxy Rice Starches.

Mild alkali treatment can potentially be developed as a greener alternative to the traditional alkali treatment of starch, but the effect of mild alkali on starch is still understudied. Normal and waxy rice starches were subjected to mild alkali combined with hydrothermal treatment to investigate their changes in physicochemical properties. After mild alkali treatment, the protein content of normal and waxy rice starches decreased from 0.76% to 0.23% and from 0.89% to 0.23%, respectively. Mild alkali treatment decreased gelatinization temperature but increased the swelling power and solubility of both starches. Mild alkali treatment also increased the gelatinization enthalpy of waxy rice starch from 20.01 J/g to 25.04 J/g. Mild alkali treatment at room temperature increased the pasting viscosities of both normal and waxy rice starches, whereas at high temperature, it decreased pasting viscosities during hydrothermal treatment. Alkali treatment significantly changed the properties of normal and waxy rice starch by the ionization of hydroxyl groups and the removal of starch granule-associated proteins. Hydrothermal conditions promoted the effect of alkali. The combination of hydrothermal and alkali treatment led to greater changes in starch properties.

Microbial production of an aromatic homopolyester

We report the development of a metabolically engineered bacterium for the fermentative production of polyesters containing aromatic side chains, serving as sustainable alternatives to petroleum-based plastics. A metabolic pathway was constructed in an Escherichia coli strain to produce poly[d-phenyllactate(PhLA)], followed by three strategies to enhance polymer production. First, polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were introduced to increase the polymer accumulation. Next, metabolic engineering was performed to redirect the metabolic flux toward PhLA. Furthermore, PHA synthase was engineered based on in silico simulation results to enhance the polymerization of PhLA. The final strain was capable of producing 12.3 g/l of poly(PhLA), marking it the first bio-based process for producing an aromatic homopolyester. Additional heterologous gene introductions led to the high level production of poly(3-hydroxybutyrate-co-11.7 mol% PhLA) copolymer (61.4 g/l). The strategies described here will be useful for the bio-based production of aromatic polyesters from renewable resources.

Mild alkali treatment alters structure and properties of maize starch: The potential role of alkali in starch chemical modification

Normal and waxy maize starches were treated with mild alkali treatment (pH 8.5, 9.9, 11.3) in two temperature-time combinations (25 °C for 1 h and 50 °C for 18 h) to investigate the effect on starch structure and properties. Mild alkali treatment partly removed the starch granule-associated proteins and lipids of normal (from 0.31 % to 0.24 % and from 0.77 % to 0.55 %, respectively) and waxy maize starches (from 0.22 % to 0.18 % and from 0.24 % to 0.15 %, respectively). Gelatinization enthalpy of waxy maize starch increased with alkali treatment from 16.20 J·g−1 to 21.95 J·g−1, indicating that amylopectin (AP) rearrangement and AP-AP double helices formation might occur. But amylose could inhibit these effects by restricting mobility of amylopectin, and no such changes occurred for normal maize starch. Alkali treatment decreased gelatinization temperature and increased peak and final viscosity. Alkali treatment decreased trough viscosity and increased setback of normal maize starch. The hydrothermal treatment promoted the effect of alkali, attributed to the more rapid molecular motion at higher temperature. Normal and waxy starches showed different changes after alkali treatment, indicating that amylose played an important role in controlling the effect of alkali and hydrothermal treatment, primarily as an obstructer of amylopectin rearrangement in mild alkali treatment.

High-throughput tagging of endogenous loci for rapid characterization of protein function.

To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.

Potential relation between starch granule-associated proteins and retrogradation properties of buckwheat starch

To elucidate the effect of starch granule-associated proteins (SGAPs) on retrogradation properties of buckwheat starch, the retrogradation properties of Tartary buckwheat starch (TBS) and common buckwheat starch (CBS) before and after removal of SGAPs were systematically investigated, with wheat starch (WS) as reference. A significant decrease in gel strength of starches and density of starch aggregates were observed after removing SGAPs. The results were in line with the changes in retrogradation enthalpy of starches and short-range ordered structure of starch aggregates. After removing SGAPs, the retrogradation enthalpy of TBS decreased from 4.16 J/g to 3.74 J/g, CBS decreased from 4.05 J/g to 3.35 J/g and WS decreased from 3.27 J/g to 2.81 J/g, respectively. Taken together the results of LF-NMR, FTIR and rheological analysis, it can be concluded that SGAPs could promote the hydrogen bond interactions between starch molecules by competitively binding with water molecules, enhancing the rearrangement of starch molecules and forming a more ordered structure. Overall, the study suggested that the presence of SGAPs could enhanced the interaction between starch molecules chains, thus accelerated the retrogradation process. The research results provide more information about SGAPs in buckwheat starch and support further study for manipulation of starch properties.

Insight into the promoted recrystallization and water distribution of bread by removing starch granule - surface and - associated proteins during storage

The influence of starch granule surface proteins (SGSPs) and starch granule-associated proteins (SGAPs) on bread retrogradation was investigated in a reconstituted dough system. The removal of both SGSPs and SGAPs resulted in poor bread qualities, decreasing specific volume and crumb porosity, leading to more baking loss and compact crumb structure. Particularly, removing SGSPs was effective in promoting the bread retrogradation. After 7 days of storage, the hardness of bread without SGSPs showed an increase of 353.34 g than the bread without SGAPs. Proton population and relaxation times exhibited that the absence of SGSPs significantly decreased the content of bound water from 11.51 % to 7.03 %, indicating lower water-holding capacity due to the loosen gelling structure. Compared to the control group, bread without SGSPs accelerated the starch recrystallinity by a reduction in soluble starch content, thereby increasing the retrogradation enthalpy and relative crystallinity through promoting the molecular reassociation in starch.

Computational Analysis of the Tripartite Interaction of Phasins (PhaP4 and 5)-Sigma Factor (σ24)-DNA of Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 produces PHB, which is covered by granule-associated proteins (GAPs). Phasins are the main GAPs. Previous studies have shown phasins can regulate PHB synthesis. When A. brasilense grows under stress conditions, it uses sigma factors to transcribe genes for survival. One of these factors is the σ24 factor. This study determined the possible interaction between phasins and the σ24 factor or phasin-σ24 factor complex and DNA. Three-dimensional structures of phasins and σ24 factor structures were predicted using the I-TASSER and SWISS-Model servers, respectively. Subsequently, a molecular docking between phasins and the σ24 factor was performed using the ClusPro 2.0 server, followed by molecular docking between protein complexes and DNA using the HDOCK server. Evaluation of the types of ligand-receptor interactions was performed using the BIOVIA Discovery Visualizer for three-dimensional diagrams, as well as the LigPlot server to obtain bi-dimensional diagrams. The results showed the phasins (Pha4Abs7 or Pha5Abs7)-σ24 factor complex was bound near the -35 box of the promoter region of the phaC gene. However, in the individual interaction of PhaP5Abs7 and the σ24 factor, with DNA, both proteins were bound to the -35 box. This did not occur with PhaP4Abs7, which was bound to the -10 box. This change could affect the transcription level of the phaC gene and possibly affect PHB synthesis.

Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

The Influence of Starch Granule-Associated Protein Removal on Starch Properties and Retrogradation

The Influence of Starch Granule-Associated Protein Removal on Starch Properties and Retrogradation

Application of PHA surface binding proteins of alkali-tolerant Bacillus as surfactants.

Amphiphilic protein has lipophilic and hydrophilic domains, displaying the potential for development as a biosurfactant. The polyhydroxyalkanoate (PHA) surface binding protein derived from Bacillus is a type of protein that has not been studied for its emulsifying properties. In this study, PHA granule-associated protein (PhaP), PHA regulatory protein (PhaQ), and PHA synthase subunit (PhaR) derived from an alkali-tolerant PHA-producing Bacillus cereus HBL-AI were found and heterologously expressed in E. coli and purified to investigate their application as biosurfactants. It showed that the emulsification ability and stability of three amphiphilic proteins were higher than those of widely used chemical surfactants in diesel oil, vegetable oil, and lubricating oil. In particular, the PhaQ protein studied for the first time can form a stable emulsion layer in vegetable oil at a lower concentration (50µg/mL), which greatly reduced the amount of protein used in emulsification. This clearly demonstrated that the PHA-binding protein of HBL-AI can be well applied as an environmentally friendly biosurfactants.

Long non-coding RNA SNHG8 drives stress granule formation in tauopathies

Tauopathies are a heterogenous group of neurodegenerative disorders characterized by tau aggregation in the brain. In a subset of tauopathies, rare mutations in the MAPT gene, which encodes the tau protein, are sufficient to cause disease; however, the events downstream of MAPT mutations are poorly understood. Here, we investigate the role of long non-coding RNAs (lncRNAs), transcripts >200 nucleotides with low/no coding potential that regulate transcription and translation, and their role in tauopathy. Using stem cell derived neurons from patients carrying a MAPT p.P301L, IVS10 + 16, or p.R406W mutation and CRISPR-corrected isogenic controls, we identified transcriptomic changes that occur as a function of the MAPT mutant allele. We identified 15 lncRNAs that were commonly differentially expressed across the three MAPT mutations. The commonly differentially expressed lncRNAs interact with RNA-binding proteins that regulate stress granule formation. Among these lncRNAs, SNHG8 was significantly reduced in a mouse model of tauopathy and in FTLD-tau, progressive supranuclear palsy, and Alzheimer’s disease brains. We show that SNHG8 interacts with tau and stress granule-associated RNA-binding protein TIA1. Overexpression of mutant tau in vitro is sufficient to reduce SNHG8 expression and induce stress granule formation. Rescuing SNHG8 expression leads to reduced stress granule formation and reduced TIA1 levels in immortalized cells and in MAPT mutant neurons, suggesting that dysregulation of this non-coding RNA is a causal factor driving stress granule formation via TIA1 in tauopathies.

USP7- and PRMT5-dependent G3BP2 stabilization drives de novo lipogenesis and tumorigenesis of HNSC

GTPase-activating protein-binding protein 2 (G3BP2) is a key stress granule-associated RNA-binding protein responsible for the formation of stress granules (SGs). Hyperactivation of G3BP2 is associated with various pathological conditions, especially cancers. Emerging evidence indicates that post-translational modifications (PTMs) play critical roles in gene transcription, integrate metabolism and immune surveillance. However, how PTMs directly regulate G3BP2 activity is lacking. Here, our analyses identify a novel mechanism that PRMT5-mediated G3BP2-R468me2 enhances the binding to deubiquitinase USP7, which ensures the deubiquitination and stabilization of G3BP2. Mechanistically, USP7- and PRMT5-dependent G3BP2 stabilization consequently guarantee robust ACLY activation, which thereby stimulating de novo lipogenesis and tumorigenesis. More importantly, USP7-induced G3BP2 deubiquitination is attenuated by PRMT5 depletion or inhibition. PRMT5-activity dependent methylation of G3BP2 is required for its deubiquitination and stabilization by USP7. Consistently, G3BP2, PRMT5 and G3BP2 R468me2 protein levels were found positively correlated in clinical patients and associated with poor prognosis. Altogether, these data suggest that PRMT5-USP7-G3BP2 regulatory axis serves as a lipid metabolism reprogramming mechanism in tumorigenesis, and unveil a promising therapeutic target in the metabolic treatment of head and neck squamous carcinoma.

Elucidating the mechanism of polyphosphate granule consolidation in bacteria.

Synthesis of polyphosphate (polyP), an ancient anionic biopolymer composed of phosphoryl groups, is universally conserved in the kingdom of life. PolyP has been implicated in promoting fitness under starvation conditions in diverse bacteria, and it has been associated with pleiotropic effects on biofilm formation, motility, cell cycle and oxidative stress resistance. The molecular mechanisms underpinning these effects have traditionally been difficult to explain. In many bacteria, polyP chains come together to form membrane-less superstructures, termed polyP granules. Some bacterial species regulate the organization of these granules. Despite the critical role of polyP in starvation fitness, the composition of these structures and the mechanism(s) underpinning their organization are poorly understood. Our previous work identified polyP-granule associated proteins in opportunistic human pathogen Pseudomonas aeruginosa. However, the mechanism(s) of polyP granule maturation and subcellular organization are not well understood. To understand the fundamental properties of polyP granules, we use in vitro reconstitution to study a simplified multicomponent system of polyP, inorganic multivalent cations, the granule-associated protein AlgP, and DNA. Using light and fluorescence microscopy, our preliminary results suggest that these ternary systems can undergo heterotypic phase transitions. Additionally, the material properties of these condensates are tunable and sensitive to environmental conditions. Interestingly, we also observe that these condensates exhibit unique sub-structure. These studies will inform future efforts to understand the basis of polyP granule composition and consolidation, as well as its functional significance to cellular fitness during starvation stress in diverse bacteria.

STRESS granule-associated RNA-binding protein CAPRIN1 drives cancer progression and regulates treatment response in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a common malignancy of the head and neck that is mainly diagnosed in southern China and Southeast Asia, with a strong etiological link to Epstein‒Barr virus infection. Those with advanced-stage disease have a significantly worse prognosis. There is an urgent need to identify novel therapeutic targets for the recurrent or metastatic nasopharyngeal carcinoma. With a particular focus on Cell Cycle Associated Protein 1 (CAPRIN1), one of the important RNA-binding proteints associated with stress granule formation, we used RT‒qPCR and immunohistochemistry to validate CAPRIN1 expression in NPC tissues and cell lines. Further, CAPRIN1 expression was knocked down using siRNA, and the effect on cell proliferation and migration was systematically assessed by in vitro assays. As a result, we demonstrated that CAPRIN1 was elevated in NPC compared to adjacent normal tissues. Knockdown of CAPRIN1 in NPC cells inhibited proliferation and migration, involving the regulation of cell cycle protein CCND2 and EMT signaling, respectively. Notably, we found that CAPRIN1 knockdown promoted cell apoptosis by regulation of the expression of apoptosis-related proteins cleaved-PARP and cleaved-Caspase3. Knockdown of CAPRIN1 increased NPC cell sensitivity to rapamycin, and increased NPC cell sensitivity to cisplatin and to X-rays. In conclusion, CAPRIN1 might drive NPC proliferation, regulate cell cycle and apoptosis, and affect tumor cell response to anti-cancer agents and X-ray irradiation. CAPRIN1 might serve as a potential target for NPC.

Freezing-induced loss of wheat starch granule-associated proteins affected dough quality: From water distribution, rheological properties, microstructure, and gluten development

This study aimed to explore the effect of freezing/thawing (F/T) treatment on starch granule-associated proteins (SGAPs) and its mechanism for modifying dough quality. SDS-PAGE showed that F/T mainly induced a loss of proteins from the starch surface (SGSPs). It was found that the decrease in associated protein content increased the content of bound water from 27.04% and 18.94% to 32.40% and 27.78% in starch and reconstituted dough systems, respectively. The absence of SGSPs resulted in a rheological behavior with higher dynamic modulus and lower strain detected by frequency sweep and creep-recovery experiments. CLSM exhibited a local aggregation of the gluten network and more starch granules dissociated. The glutenin macropolymers (GMP) content increased from 76.45% to 83.31% while the values of fluorescence intensity and free sulfhydryl (SH) decreased to 2821 and 1.85 μmol/g, respectively. These results suggested that SGSPs acted as a contact site in the “starch-gluten” matrix, facilitating macromolecular adhesion.

Co-Culture of Halotolerant Bacteria to Produce Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Using Sewage Wastewater Substrate.

The focus of the current study was the use of sewage wastewater to obtain PHA from a co-culture to produce a sustainable polymer. Two halotolerant bacteria, Bacillus halotolerans 14SM (MZ801771) and Bacillus aryabhattai WK31 (MT453992), were grown in a consortium to produce PHA. Sewage wastewater (SWW) was used to produce PHA, and glucose was used as a reference substrate to compare the growth and PHA production parameters. Both bacterial strains produced PHA in monoculture, but a copolymer was obtained when the co-cultures were used. The co-culture accumulated a maximum of 54% after 24 h of incubation in 10% SWW. The intracellular granules indicated the presence of nucleation sites for granule initiation. The average granule size was recorded to be 231 nm; micrographs also indicated the presence of extracellular polymers and granule-associated proteins. Fourier transform infrared spectroscopy (FTIR) analysis of the polymer produced by the consortium showed a significant peak at 1731 cm-1, representing the C=O group. FTIR also presented peaks in the region of 2800 cm-1 to 2900 cm-1, indicating C-C stretching. Proton nuclear magnetic resonance (1HNMR) of the pure polymer indicated chemical shifts resulting from the proton of hydroxy valerate and hydroxybutyrate, confirming the production of poly(3-hydroxybutyrate-co-3-hydroxy valerate) (P3HBV). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that the copolymer was biocompatible, even at a high concentration of 5000 µg mL-1. The results of this study show that bacterial strains WK31 and 14SM can be used to synthesize a copolymer of butyrate and valerate using the volatile fatty acids present in the SWW, such as propionic acid or pentanoic acid. P3HBV can also be used to provide an extracellular matrix for cell-line growth without causing any cytotoxic effects.