The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous ...recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
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•DSS1 works in conjunction with BRCA2 to facilitate RPA-RAD51 exchange on ssDNA•DSS1 interacts with RPA via its solvent-exposed acidic loop•DSS1 binds the RPA70 subunit of RPA and attenuates ssDNA binding by it•DSS1 could also function as a nucleic acid mimic in other biological processes
How RPA is displaced from DNA to enable RAD51 recombinase loading in homologous recombination (HR) is not known. Zhao et al. show that DSS1 targets RPA and mimics DNA to facilitate RPA displacement. This identifies DSS1 as an essential component of a HR mediator complex with the tumor suppressor BRCA2.
Advances in amyloid research rely on improved access to the β-amyloid peptide, Aβ. N-Terminal methionine-extended Aβ, Aβ(M1–42), is a readily expressed and widely used form of Aβ with properties ...comparable to those of the natural Aβ(1–42) peptide. Expression of Aβ(M1–42) is simple to execute and avoids an expensive and often difficult enzymatic cleavage step associated with expression and isolation of Aβ(1–42). This paper reports an efficient method for the expression and purification of Aβ(M1–42) and 15N-labeled Aβ(M1–42). This method affords the pure peptide at ∼19 mg/L of bacterial culture through simple and inexpensive steps in 3 days. This paper also reports a simple method for the construction of recombinant plasmids and the expression and purification of Aβ(M1–42) peptides containing familial mutations. We anticipate that these methods will enable experiments that would otherwise be hindered by insufficient access to Aβ.
Synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are defined by the presence of intracellular alpha-synuclein aggregates in ...neurons and/or oligodendrocytes. In addition, post mortem tissue analysis revealed profound changes in microglial morphology, indicating microglial activation and neuroinflammation. Thus, alpha-synuclein may directly activate microglia, leading to increased production of key pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), which in turn modulates the disease progression. The distinct alpha-synuclein species, which mediates the activation of microglia, is not well defined. We hypothesized that microglial activation depends on a specific aggregation state of alpha-synuclein. Here, we show that primarily human fibrillar alpha-synuclein increased the production and secretion of pro-inflammatory cytokines by microglial BV2 cells compared to monomeric and oligomeric alpha-synuclein. BV2 cells also preferentially phagocytosed fibrillar alpha-synuclein compared to alpha-synuclein monomers and oligomers. Microglial uptake of alpha-synuclein fibrils and the consequent activation were time- and concentration-dependent. Moreover, the degree of fibrillization determined the efficiency of microglial internalization. Taken together, our study highlights the specific crosstalk of distinct alpha-synuclein species with microglial cells.
•Physico-chemical modification of alpha-synuclein leads to oligomerization and fibrillization.•Primarily fibrillar alpha-synuclein increased production and secretion of TNF-α and IL-1β in BV2 cells.•Fibrillar alpha-synuclein was efficiently internalized by BV2 cells.•Exposure to alpha-synuclein had no detrimental effects on viability of BV2 cells.
Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine ...CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13
follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.
Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, ...however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.
Protein (19)F NMR in Escherichia coli Li, Conggang; Wang, Gui-Fang; Wang, Yaqiang ...
Journal of the American Chemical Society,
2010-Jan-13, 20100113, Letnik:
132, Številka:
1
Journal Article
Recenzirano
Although overexpression and (15)N enrichment facilitate the observation of resonances from disordered proteins in Escherichia coli, (15)N enrichment alone is insufficient for detecting most globular ...proteins. Here, we explain this dichotomy and overcome the problem while extending the capability of in-cell NMR by using (19)F-labeled proteins. Resonances from small (approximately 10 kDa) globular proteins containing the amino acid analogue 3-fluoro-tyrosine can be observed in cells, but for larger proteins the (19)F resonances are broadened beyond detection. Incorporating the amino acid analogue trifluoromethyl-L-phenylalanine allows larger proteins (up to 100 kDa) to be observed in cells. We also show that site-specific structural and dynamic information about both globular and disordered proteins can be obtained inside cells by using (19)F NMR.
Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and ...mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.
Strigolactones (SLs) are phytohormones that inhibit shoot branching and function in the rhizospheric communication with symbiotic fungi and parasitic weeds. An α/β-hydrolase protein, DWARF14 (D14), ...has been recognized to be an essential component of plant SL signalling, although its precise function remains unknown. Here we present the SL-dependent interaction of D14 with a gibberellin signalling repressor SLR1 and a possible mechanism of phytohormone perception in D14-mediated SL signalling. D14 functions as a cleavage enzyme of SLs, and the cleavage reaction induces the interaction with SLR1. The crystal structure of D14 shows that 5-hydroxy-3-methylbutenolide (D-OH), which is a reaction product of SLs, is trapped in the catalytic cavity of D14 to form an altered surface. The D14 residues recognizing D-OH are critical for the SL-dependent D14-SLR1 interaction. These results provide new insight into crosstalk between gibberellin and SL signalling pathways.
We present a simple, convenient and robust protocol for expressing perdeuterated proteins in
E.
coli
BL21(DE3) cells in shaker flasks that reduces D
2
O usage tenfold and d
7
-glucose usage by ...30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD
600
of up to 10. Inducing the cells with isopropyl β-
d
-1-thiogalactopyranoside at an OD
600
of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD
600
between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of
E.
coli
from H
2
O-based to D
2
O-based medium is also key for ensuring high levels of protein expression in D
2
O. We find that a simple three-step adaptation approach—Luria–Bertani (LB) medium in H
2
O to LB in D
2
O to modified-M9 medium in D
2
O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.
Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of ...both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein-protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.
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