The modulation of protein–protein interactions (PPIs) has been recognized as one of the most challenging tasks in drug discovery. While their systematic development has long been considered as ...intractable, this view has changed over the last years, with the first drug candidates undergoing clinical studies. To date, the vast majority of PPI modulators are interaction inhibitors. However, in many biological contexts a prolonged lifespan of a PPI might be desirable, calling for the complementary approach of PPI stabilization. In fact, nature offers impressive examples of this concept and some PPI‐stabilizing natural products have already found application as important drugs. Moreover, directed small‐molecule stabilization has recently been demonstrated. Therefore, it is time to take a closer look at the constructive side of modulating PPIs.
Doing it the other way round: The modulation of protein–protein interactions (PPIs) by small molecules has become increasingly popular over the last few decades. However, “modulation” has mainly been perceived as “inhibition” of protein–protein interactions, omitting the complementary strategy of stabilizing such macromolecular complexes. This Minireview highlights amazing examples and the potential of this constructive side of modulating PPIs.
During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. ...By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.
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•VCP/p97 extracts sterically trapped Ku80 after NHEJ•VCP/p97 targets Ku modified with K48-linked ubiquitin chains•VCP/p97 is also essential for DNA repair pathway choice
During DNA double-strand break repair by non-homologous end joining, the ring-shaped Ku heterodimer becomes sterically trapped on DNA and interferes with chromatin functionality. van den Boom et al. use a combination of in vitro and cell-based approaches to reveal that the AAA+-type ATPase VCP/p97 drives Ku extraction after DNA repair.
Protein quality control involves sensing and treatment of defective or incomplete protein structures. Misfolded or mislocalized proteins trigger dedicated signal transduction cascades that upregulate ...the production of protein quality-control factors. Corresponding proteases and chaperones either degrade or repair damaged proteins, thereby reducing the level of aggregation-prone molecules. Because the periplasm of gram-negative bacteria is particularly exposed to environmental changes and respective protein-folding stresses connected with the presence of detergents, low or high osmolarity of the medium, elevated temperatures, and the host's immune response, fine-tuned protein quality control systems are essential for survival under these unfavorable conditions. This review discusses recent advances in the identification and characterization of the key cellular factors and the emerging general principles of the underlying molecular mechanisms.
The objective of this preliminary study was to explore long-term changes in neurobehavioral parameters, brain morphology and electroencephalography of sepsis patients who received intensive care ...compared to non-septic intensive care unit (ICU) patients.
Two-centre follow-up study 6-24 months after discharge from hospital using published norms and existing databases of healthy controls for comparison. Patients included 25 septic and 19 non-septic ICU survivors who were recruited from two ICUs of a university and community hospital. Measurements used include brain morphology, standard electroencephalography, cognition and psychiatric health and health-related quality of life.
Sepsis survivors showed cognitive deficits in verbal learning and memory and had a significant reduction of left hippocampal volume compared to healthy controls. Moreover, sepsis and to some extent non-septic ICU patients had more low-frequency activity in the EEG indicating unspecific brain dysfunction. No differences were found in health-related quality of life, psychological functioning or depressive symptoms, and depression could be ruled out as a confounding factor.
This study demonstrates permanent cognitive impairment in several domains in both septic and non-septic ICU survivors and unspecific brain dysfunction. In the sepsis group, left-sided hippocampal atrophy was found compared to healthy controls. Further study is needed to clarify what contribution sepsis and other factors at the ICU make to these outcomes. Specific neuroprotective therapies are warranted to prevent persisting brain changes in ICU patients.
Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic ...release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted β-galactosidase 1 (BGAL1) of
promotes hydrolytic elicitor release and acts in immunity against pathogenic
strains only when they carry a terminal modified viosamine (mVio) in the flagellin
-glycan. In counter defense,
pathovars evade host immunity by using BGAL1-resistant
-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.
Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use ...dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved serine protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed.
Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies ...demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.
Abstract
The systematic stabilization of protein–protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current ...lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.
Radiation-induced normal tissue toxicity is closely linked to endothelial cell (EC) damage and dysfunction (acute effects). However, the underlying mechanisms of radiation-induced adverse late ...effects with respect to the vascular compartment remain elusive, and no causative radioprotective treatment is available to date.
The importance of injury to EC for radiation-induced late toxicity in lungs after whole thorax irradiation (WTI) was investigated using a mouse model of radiation-induced pneumopathy. We show that WTI induces EC loss as long-term complication, which is accompanied by the development of fibrosis. Adoptive transfer of mesenchymal stem cells (MSCs) either derived from bone marrow or aorta (vascular wall-resident MSCs) in the early phase after irradiation limited the radiation-induced EC loss and fibrosis progression. Furthermore, MSC-derived culture supernatants rescued the radiation-induced reduction in viability and long-term survival of cultured lung EC. We further identified the antioxidant enzyme superoxide dismutase 1 (SOD1) as a MSC-secreted factor. Importantly, MSC treatment restored the radiation-induced reduction of SOD1 levels after WTI. A similar protective effect was achieved by using the SOD-mimetic EUK134, suggesting that MSC-derived SOD1 is involved in the protective action of MSC, presumably through paracrine signaling.
In this study, we explored the therapeutic potential of MSC therapy to prevent radiation-induced EC loss (late effect) and identified the protective mechanisms of MSC action.
Adoptive transfer of MSCs early after irradiation counteracts radiation-induced vascular damage and EC loss as late adverse effects. The high activity of vascular wall-derived MSCs for radioprotection may be due to their tissue-specific action. Antioxid. Redox Signal. 26, 563-582.
Spirochaetes are frequently detected in anoxic hydrocarbon- and organohalide-polluted groundwater, but their role in such ecosystems has remained unclear. To address this, we studied a ...sulfate-reducing, naphthalene-degrading enrichment culture, mainly comprising the sulfate reducer Desulfobacterium N47 and the rod-shaped Spirochete Rectinema cohabitans HM. Genome sequencing and proteome analysis suggested that the Spirochete is an obligate fermenter that catabolizes proteins and carbohydrates, resulting in acetate, ethanol, and molecular hydrogen (H
) production. Physiological experiments inferred that hydrogen is an important link between the two bacteria in the enrichment culture, with H
derived from fermentation by R. cohabitans used as reductant for sulfate reduction by Desulfobacterium N47. Differential proteomics and physiological experiments showed that R. cohabitans utilizes biomass (proteins and carbohydrates) released from dead cells of Desulfobacterium N47. Further comparative and community genome analyses indicated that other Rectinema phylotypes are widespread in contaminated environments and may perform a hydrogenogenic fermentative lifestyle similar to R. cohabitans. Together, these findings indicate that environmental Spirochaetes scavenge detrital biomass and in turn drive necromass recycling at anoxic hydrocarbon-contaminated sites and potentially other habitats.
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