Aggregation Hotspots Research Articles

Spatial epidemiological analysis based on township scale and analysis of influencing factors of pulmonary tuberculosis cure of Changshu city from 2015 to 2022.

This study aimed to enhance the prevention and control of pulmonary tuberculosis (PTB) and provide more effective and accurate methods in Changshu City. The PTB patients' information came from the China Information System for Disease Control and Prevention (CISDCP). The demographic data for Changshu city and towns came from the Suzhou Statistical Yearbook and the LandScan platform. ArcGIS was used for global spatial autocorrelation analysis and local spatial autocorrelation analysis. Univariate logistic regression and multivariate logistic regression were used to analyze the influencing factors of cured PTB patients. The receiver operating characteristic (ROC) curve and decision curve analysis (DCA) were used to analyze the predictive efficacy and clinical benefit of the indicators. XGBoost analysis was performed to explore the feature importance of key metrics for PTB outcome. A total of 3943 PTB cases were included. The annual incidence rate of new PTB in Changshu city was 27.081 per 100,000. Changshu High-tech Industrial Development Zone in Jiangsu Province and Shajiabang town were the high-high aggregation areas and hot spot areas. Diagnosis delay, TB strain types, and drug sensitivity were independent predictors of the cure of new PTB patients. The central and southern areas of Changshu were the high-high cluster areas and hot spots for PTB. Shorter diagnosis delay days and mycobacterium tuberculosis (MTB) promote the cure of tuberculosis, while drug sensitivity was a risk factor for its cure.

Development and characterization of novel anti-acetylated tau monoclonal antibodies to probe pathogenic tau species in Alzheimer’s disease

Tauopathies, including Alzheimer’s disease (AD), are a class of neurodegenerative diseases characterized by the presence of insoluble tau inclusions. Tau phosphorylation has traditionally been viewed as the dominant post-translational modification (PTM) controlling tau function and pathogenesis in tauopathies. However, we and others have identified tau acetylation as a primary PTM regulating both normal tau function as well as abnormal pathogenic features including aggregation. Prior work showed robust tau acetylation in aggregation hotspots located within the 2nd and 3rd repeat regions of tau (residues K280 and K311) in tauopathy brains, including AD, compared to non-tauopathy controls. By screening thousands of hybridoma clones, we generated site-specific and modification-specific monoclonal antibodies targeting acetylated tau at residues K280 or K311. To validate these antibodies in a bona fide neuronal system, we targeted the acetyltransferase CBP to the cytoplasm of neurons to promote tau acetylation. Several antibody clones specifically detected CBP-acetylated tau and co-localized with ac-tau in neurons. Additionally, our lead optimal anti-acetylated-tau monoclonal antibodies detected robust tau pathology in tangles and neuritic plaques of human AD brains. Given the now emerging interest in acetylated tau as critical regulator of tau functions, these sensitive and highly specific tools will allow us to further unravel the tau PTM code and, importantly, could be deployed as diagnostic or disease-modifying agents.

56P Mutational screening of distal myopathy gene ACTN2 reveals an aggregation hot-spot in the actin-binding domain

56P Mutational screening of distal myopathy gene ACTN2 reveals an aggregation hot-spot in the actin-binding domain

Furosemide Derails Human Lysozyme Fibrillation by Interacting with Aggregation Hot Spots: A Biophysical Comprehension.

Kidney-associated human lysozyme amyloidosis leads to renal impairments;thus, patients are often prescribed furosemide. Based on this fact, the effect of furosemide on induced human lysozyme fibrillation, in vitro, is evaluated by spectroscopic, calorimetric, computational, and cellular-based assays/methods. Results show that furosemide increases the lag phase and decreases the apparent rate of aggregation of human lysozyme, thereby decelerating the nucleation phase and amyloid fibril formation, as confirmed by the decrease in the level of Thioflavin-T fluorescence. Fewer entities of hydrodynamic radii of ∼171 nm instead of amyloid fibrils (∼412 nm) are detected in human lysozyme in the presence of furosemide by dynamic light scattering. Moreover, furosemide decreases the extent of conversion of the α/β structure of human lysozyme into a predominant β-sheet. The isothermal titration calorimetry established that furosemide forms a complex with human lysozyme, which was also confirmed through fluorescence quenching and computational studies. Also, human lysozyme lytic activity is inhibited competitively by furosemide due to the involvement of amino acid residues of the active site in catalysis, as well as complex formation. Conclusively, furosemide interacts with Gln58, Ile59, Asn60, Ala108, and Trp109 of aggregation-prone regions 2 and 4 of human lysozyme, thereby masking its sites of aggregation and generating only lower-order entities that are less toxic to red blood cells than the fibrils. Thus, furosemide slows the progression of amyloid fibrillation in human lysozyme.

Spatiotemporal variations in reef manta ray (Mobula alfredi) residency at a remote meso‐scale habitat and its importance in future spatial planning

Abstract The Chagos Archipelago's vast no‐take marine protected area (MPA, 640,000 km2) provides refuge for elasmobranchs facing unsustainable depletion by fisheries. Nonetheless, illegal, unreported and unregulated (IUU) fishing poses a substantial threat, and potential future changes to the use of the MPA could render elasmobranchs increasingly vulnerable to exploitation, putting geographically isolated populations, such as reef manta rays (Mobula alfredi) at risk of local extinction. Therefore, the species' long‐term movements and habitat use must be identified to help prioritize current enforcement activity and inform future spatial planning. Passive acoustic telemetry and modelled environmental data were used to investigate variations in 42 tagged M. alfredi utilization of a meso‐scale aggregation hotspot, Egmont Atoll, between 2019 and 2022. Mobula alfredi displayed the highest levels of residency ever reported (77%), with prolonged absences (>2 months) limited to seven individuals. Egmont atoll was used year‐round, with activity peaks during the southeast monsoon (April – November), particularly at sites on the southwest, while sites on the northwest were predominately frequented in the northwest monsoon (December–March). Tags were most likely to be detected when the Indian Ocean Dipole (IOD) was in a positive phase with a greater mixed layer depth, associated with a depression of chlorophyll α levels in the Indian Ocean. Thus, M. alfredi may be particularly reliant on Egmont Atoll, where they are predominantly observed feeding, when prey resources are limited elsewhere. In a region where the threat of fisheries is of increasing concern, the identification of crucial M. alfredi habitats is essential for conservation management planning. Given the significant role of Egmont Atoll for the local population, regular IUU enforcement patrols are crucial, particularly during the southeast monsoon. Any future changes to the MPA should prioritize preserving and actively enforcing no‐take regulations at Egmont Atoll.

Identification of potential aggregation hotspots on Aβ42 fibrils blocked by the anti-amyloid chaperone-like BRICHOS domain

Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer’s disease (AD). The surface of AD-associated amyloid-β peptide (Aβ) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle. Here, we identify a site on the Aβ42 fibril surface, consisting of three C-terminal β-strands and particularly the solvent-exposed β-strand stretching from residues 26–28, which is efficiently sensed by a designed variant of Bri2 BRICHOS. Remarkably, while only a low amount of BRICHOS binds to Aβ42 fibrils, fibril-catalyzed nucleation processes are effectively prevented, suggesting that the identified site acts as a catalytic aggregation hotspot, which can specifically be blocked by BRICHOS. Hence, these findings provide an understanding how toxic nucleation events can be targeted by molecular chaperones.

Study on the spatial correlation effects and influencing factors of carbon emissions from the electricity industry: a fresh evidence from China.

Carbon emissions from the electricity industry (CEEI) account for a large proportion of China's total carbon emissions, and it is important to study the spatial correlation between CEEI and the influencing factors to promote cross-regional synergistic emission reduction and low-carbon development of the power system. In this paper, the quasi-input-output (QIO) model is applied to assess the transfer of carbon emissions generated by electricity trading based on the consideration of electricity carbon transfer, and the exploratory spatial data analysis (ESDA) method is applied to analyze the spatial correlation effect of carbon emissions from China's electric power sector from 2001 to 2020, analyzes its distribution pattern in both spatial and temporal dimensions, and applies the improved logarithmic mean Divisia index (LMDI) two-stage decomposition model to decompose the changes in CEEI into 11 influencing factors from the perspective of the whole industrial chain of power production, transmission, trade, and consumption. The research results show that (1) the spatial distribution of CEEI has obvious unevenness and aggregation characteristics, with high-high aggregation areas and hot spot aggregation areas generally concentrated in the North China Power Grid and the East China Power Grid, but the aggregation trend is gradually decreasing, while low-low aggregation areas and cold spot aggregation areas are concentrated in the Northwest China Power Grid and the Central China Power Grid, but the area is very limited. (2) The direction of carbon emission diffusion in China's electricity industry is gradually transitioning from southwest-northeast to northwest-southeast, and the east-west diffusion trend is stronger than the north-south diffusion trend and carbon emissions are gradually shifting to the northwest grid. (3) The total amount of electricity production is the most influential factor in the change of CEEI, driving the cumulative growth of CEEI by 4495.34 Mt, followed by GDP per capita and electricity consumption intensity. Coal consumption for power generation, the share of thermal power, and net electricity exports were the main factors inhibiting the increase in carbon emissions from the power sector, with cumulative contributions of -797.74 Mt, -619.99 Mt, and -47.76 Mt, respectively.

Shape- and polymer-considered simulation to unravel the estuarine microplastics fate

Environmental microplastics (MPs) constitute various sizes, polymers, and shape components. In estuaries, such differences are related to the reliability of assessing the seaward fate of MPs, aggregation hotspots, and ecological risks. This study sets the MP particle mass gradient using the shape factor and size probability density function to categorically estimate the MP load in the surface layer of the Yangtze River Estuary (YRE), which is the largest contributor of plastics to the sea. During the high plastic input period in July, the optimized estimated MP load through the surface layer of the YRE was 9766 kg/month, which was overestimated by 821 kg/month based on the empirical average particle mass. While tracking MP transport classified by shape and polymer type, the resuspension of MPs that accumulate in the intertidal zone cannot be neglected. The average relative error of the simulation was as low as 19.6% after including the abovementioned factors. Finally, the simulation results of the sensitive regions were extracted to assess the new MP risk index, which considers shape, abundance, and polymer type. By introducing these essential tools, this study helps to understand the fate of riverine MPs entering estuaries, where valuable opportunities for removing MPs exist before they spread to the oceans.

Intrinsic disorder in PRAME and its role in uveal melanoma

IntroductionThe PReferentially expressed Antigen in MElanoma (PRAME) protein has been shown to be an independent biomarker for increased risk of metastasis in Class 1 uveal melanomas (UM). Intrinsically disordered proteins and regions of proteins (IDPs/IDPRs) are proteins that do not have a well-defined three-dimensional structure and have been linked to neoplastic development. Our study aimed to evaluate the presence of intrinsic disorder in PRAME and the role these structureless regions have in PRAME( +) Class 1 UM.MethodsA bioinformatics study to characterize PRAME’s propensity for the intrinsic disorder. We first used the AlphaFold tool to qualitatively assess the protein structure of PRAME. Then we used the Compositional Profiler and a set of per-residue intrinsic disorder predictors to quantify the intrinsic disorder. The Database of Disordered Protein Prediction (D2P2) platform, IUPred, FuzDrop, fIDPnn, AUCpred, SPOT-Disorder2, and metapredict V2 allowed us to evaluate the potential functional disorder of PRAME. Additionally, we used the Search Tool for the Retrieval of Interacting Genes (STRING) to analyze PRAME's potential interactions with other proteins.ResultsOur structural analysis showed that PRAME contains intrinsically disordered protein regions (IDPRs), which are structureless and flexible. We found that PRAME is significantly enriched with serine (p-value < 0.05), a disorder-promoting amino acid. PRAME was found to have an average disorder score of 16.49% (i.e., moderately disordered) across six per-residue intrinsic disorder predictors. Our IUPred analysis revealed the presence of disorder-to-order transition (DOT) regions in PRAME near the C-terminus of the protein (residues 475–509). The D2P2 platform predicted a region from approximately 140 and 175 to be highly concentrated with post-translational modifications (PTMs). FuzDrop predicted the PTM hot spot of PRAME to be a droplet-promoting region and an aggregation hotspot. Finally, our analysis using the STRING tool revealed that PRAME has significantly more interactions with other proteins than expected for randomly selected proteins of the same size, with the ability to interact with 84 different partners (STRING analysis result: p-value < 1.0 × 10–16; model confidence: 0.400).ConclusionOur study revealed that PRAME has IDPRs that are possibly linked to its functionality in the context of Class 1 UM. The regions of functionality (i.e., DOT regions, PTM sites, droplet-promoting regions, and aggregation hotspots) are localized to regions of high levels of disorder. PRAME has a complex protein–protein interaction (PPI) network that may be secondary to the structureless features of the polypeptide. Our findings contribute to our understanding of UM and suggest that IDPRs and DOT regions in PRAME may be targeted in developing new therapies for this aggressive cancer.BBcyEW8tS6_KkPjyEwduzxVideo

Bacterial Lectin FimH and Its Aggregation Hot-Spots: An Alternative Strategy against Uropathogenic Escherichia coli.

Type I fimbriae are the main adhesive organelles of uropathogenic Escherichia coli (UPEC), consisting of four different subunits. Their component with the most important role in establishing bacterial infections is the FimH adhesin located at the fimbrial tip. This two-domain protein mediates adhesion to host epithelial cells through interaction with terminal mannoses on epithelial glycoproteins. Here, we propose that the amyloidogenic potential of FimH can be exploited for the development of therapeutic agents against Urinary Tract Infections (UTIs). Aggregation-prone regions (APRs) were identified via computational methods, and peptide-analogues corresponding to FimH lectin domain APRs were chemically synthesized and studied with the aid of both biophysical experimental techniques and molecular dynamic simulations. Our findings indicate that these peptide-analogues offer a promising set of antimicrobial candidate molecules since they can either interfere with the folding process of FimH or compete for the mannose-binding pocket.

Rational Design and Production of Bioactive Analogs of Recombinant Human Keratinocyte Growth Factor (rhKGF) with Reduced Aggregation Propensity.

Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).

Theoretical investigations of TTR derived aggregation-prone peptides’ potential to biochemically attenuate the amyloidogenic propensities of V30 M TTR amyloid fibrils

Transthyretin (TTR) is a cerebrospinal fluid and plasma prevalent protein implicated in heritable and sporadic amyloidosis. Numerous mutations and a wide range of phenotypes have been associated with TTR-mediated amyloidosis. Among these, V30 M is the most predominant point mutation, inculpated with familial amyloid polyneuropathy (FAP), a life-threatening autosomal dominant genetic disorder characterized by the deposition of amyloid fibrils in crucial areas. Hence, efficacious therapeutics against this detrimental disorder is warranted. Lately, several peptide-based analeptics, especially the ones that are aggregation-prone and the ones derived from aggregation hotspots of amyloidogenic proteins are being increasingly proffered against the amyloid fibrils. In the present study, as an effective precursor to in vitro investigations, we examined and assessed the therapeutic potentials of aggregation-prone peptides (APPs) derived from TTR, against V30 M TTR amyloid fibrils, computationally. Out of five experimentally corroborated APPs availed for this study, molecular dynamics simulation analysis endorses APP TAVVTN to be an effective beta-sheet breaker against V30 M TTR amyloid fibrils. Furthermore, consistent findings from various molecular trajectory analyses, residual frustration analysis and simulated thermal denaturation have indicated that APP TAVVTN could effectually relater the structural dynamics of V30 M TTR amyloid fibrils, to conformationally digress it away from its amyloidogenic propensities. Hence, based on consistent unvarying findings from numerous adept computational pipelines, APP TAVVTN could be an efficacious analeptic to therapeutically intervene and mitigate the amyloidogenic propensities of V30 M TTR amyloid fibrils, thereby ameliorating the pathological ramifications due to FAP.

Sequence-based Prediction of the Cellular Toxicity Associated with Amyloid Aggregation within Protein Condensates.

Various neurological dysfunctions are associated with cytotoxic amyloid-containing aggregates formed through the irreversible maturation of protein condensates generated by phase separation. Here, we investigate the amino acid code for this cytotoxicity using TDP-43 deep-sequencing data. Within the droplet landscape framework, we analyze the impact of mutations in the amyloid core, aggregation hot-spot, and droplet-promoting residues on TDP-43 cytotoxicity. Our analysis suggests that TDP-43 mutations associated with low cytotoxicity moderately decrease the probability of droplet formation while increasing the probability of multimodal binding. These mutations promote both ordered and disordered binding modes, thus facilitating the conversion between the droplet and amyloid states. Based on this understanding, we develop an extension of the FuzDrop method for the sequence-based prediction of the cytotoxicity of aging condensates and test it over 20,000 TDP-43 variants. Our analysis provides insight into the amino acid code that regulates the cytotoxicity associated with the maturation of liquid-like condensates into amyloid-containing aggregates, suggesting that, at least in the case of TDP-43, mutations that promote aggregation tend to decrease cytotoxicity, while those that promote droplet formation tend to increase cytotoxicity.

Rational Design of the Soluble Variant of l-Pipecolic Acid Hydroxylase using the α-Helix Rule and the Hydropathy Contradiction Rule.

The production of recombinant proteins in Escherichia coli is an important application of biotechnology. 2-Oxoglutarate-dependent l-pipecolic acid hydroxylase derived from Xenorhabdus doucetiae (XdPH) is an excellent biocatalyst that catalyzes the hydroxylation of l-pipecolic acid to produce cis-5-hydroxy-l-pipecolic acid. However, the enzyme tends to form aggregates in the E. coli expression system. Our group established two rules, namely, the “α-helix rule” and the “hydropathy contradiction rule,” to select residues to be altered for improving the heterologous recombinant production of proteins, by analyzing their primary structure. We rationally designed XdPH variants that are expressed in highly soluble and active forms in the E. coli expression system using these hotspot prediction methods, and the L142R variant showed a remarkably high soluble expression level compared to the wild-type XdPH. Further mutations were introduced into the L142R gene by site-directed mutagenesis. Moreover, the I28P/L142R and C76Y/L142R double variants displayed improved soluble expression levels compared to the single variants. These variants were also more thermostable than the wild-type XdPH. To analyze the effect of the alteration on one of the hotspots, L142 was replaced with various hydrophilic and positively charged residues. The remarkable increase in soluble protein expression caused by the alterations suggests that the decrease in the hydrophobicity of the protein surface and the enhancement of the interaction between nearby residues are important factors determining the solubility of the protein. Overall, this study demonstrated the effectiveness of our protocol in identifying aggregation hotspots for recombinant protein production and in basic biochemical research.

FuzDrop on AlphaFold: visualizing the sequence-dependent propensity of liquid-liquid phase separation and aggregation of proteins.

Many proteins perform their functions within membraneless organelles, where they form a liquid-like condensed state, also known as droplet state. The FuzDrop method predicts the probability of spontaneous liquid–liquid phase separation of proteins and provides a sequence-based score to identify the regions that promote this process. Furthermore, the FuzDrop method estimates the propensity of conversion of proteins to the amyloid state, and identifies aggregation hot-spots, which can drive the irreversible maturation of the liquid-like droplet state. These predictions can also identify mutations that can induce formation of amyloid aggregates, including those implicated in human diseases. To facilitate the interpretation of the predictions, the droplet-promoting and aggregation-promoting regions can be visualized on protein structures generated by AlphaFold. The FuzDrop server (https://fuzdrop.bio.unipd.it) thus offers insights into the complex behavior of proteins in their condensed states and facilitates the understanding of the functional relationships of proteins.

Physical and environmental drivers of oceanic manta ray Mobula birostris sightings at an aggregation site in Bahía de Banderas, Mexico

Oceanic manta raysMobula birostrisare distributed in tropical and subtropical oceans with aggregation sites typically at oceanic islands and seamounts. However, in some regions the species is found in coastal habitats close to highly productive areas. Recently, an important aggregation site for oceanic manta rays was identified along the southern coast of Bahía de Banderas, Mexico. We conducted weekly monitoring trips to the aggregation hotspots over a period of 4½ yr, recording manta sightings and collecting zooplankton samples, to investigate relationships between sightings and prey availability. We evaluated relationships between manta sightings and physical, biological, and environmental variables, finding a seasonal signal of manta occurrence with a peak in sightings around April, although there were substantial deviations from this trend in some years. Our results show effects of the El Niño Southern Oscillation on sighting rates, with more mantas during La Niña phases. We found significant relationships between manta sightings and sea surface temperature, moon phase, and tidal range. Manta sightings were negatively related to fish egg densities, positively related to copepod and cladoceran densities, and showed no relationship to euphausiid density. Mantas may be feeding on mesopelagic prey in the submarine canyon adjacent to the southern coast and basking in shallow waters through the day during thermal recovery periods. Identifying the seasonal patterns of occurrence and the environmental drivers of sightings will support the development of strategies to mitigate anthropogenic threats, which are common in this coastal population.

Charge neutralization of lysine via carbamylation reveals hidden aggregation hot-spots in tau protein flanking regions.

Tau protein is found abundantly in neurofibrillary tangles in Alzheimer's disease (AD). The longest human tau isoform (2N4R) has 44 lysine residues. Several lysine-based post-translational modifications (PTMs) such as glycation, acetylation, ubiquitination, and sumoylation have been implicated not only in AD, but also in other tauopathies. Carbamylation is one such lysine neutralizing age-related nonenzymatic PTM which can modulate the aggregation propensity of tau. In this work, we have studied the aggregation potential of lysine-rich regions of tau upon carbamylation which do not aggregate in their native form. Using an array of biophysical and microscopic analyses, such as ThT kinetic assay, fluorescence microscopy, Congo red staining, and scanning electron microscopy, we demonstrate that peptides derived from four of five such regions exhibit robust fibrillar amyloid formation. These regions are found in the N-terminal projection domain that encompasses proline-rich domain (148-153 and 223-230), repeat domain R1 (253-260), as well as fibrillary core region (368-378), and can be described as hidden aggregation hot-spots which become activated upon carbamylation. We have further compared the impact of carbamylation with acetylation on the aggregation propensity of lysine-rich peptide (254 KKVAVV259 ) using biophysical experiments and molecular dynamics simulations and deduced that carbamylation is a much stronger driver of aggregation than acetylation. Our findings may offer more insight into amyloid fibrils' interaction with hidden aggregation-prone nucleating sequences that act as hot-spots for inducing tau fibrillation.

Protein engineering of the aldoxime dehydratase from Bacillus sp. OxB-1 based on a rational sequence alignment approach

Recently, the program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced. We explored the utility of this program further and applied it for engineering of the aldoxime dehydratase from Bacillus sp. OxB-1. Towards this end, the effect of inverting the hydropathy at selected positions of the amino acid sequence on the enzymatic activity was studied leading to 60% of our constructed variants, which showed improved activity. In part, this activity increase can be rationalised by an improved heme incorporation of the variants. For example, a single mutation gave a 1.8 fold increased enzymatic activity and 30% improved absolute heme incorporation.

Optimized expression and purification of a soluble BMP2 variant based on in-silico design

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.

Potential aggregation hot spots in recombinant human keratinocyte growth factor: a computational study

The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10–30, 40–60, 61–66, 88–120, and 130–140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency. Communicated by Ramaswamy H. Sarma