Hypoxia-dependent recruitment of error-prone DNA polymerases to genome replication.

Hypoxia is common in tumors and is associated with cancer progression and drug resistance, driven, at least in part, by genetic instability. Little is known on how hypoxia affects Translesion DNA Synthesis (TLS), in which error-prone DNA polymerases bypass lesions, thereby maintaining DNA continuity at the price of increased mutations. Here we show that under acute hypoxia, PCNA monoubiquitination, a key step in TLS, and expression of error-prone DNA polymerases increased under regulation of the HIF1α transcription factor.

The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma.

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1.

Hsp90 Co-chaperones Form Plastic Genetic Networks Adapted to Client Maturation.

Heat shock protein 90 (Hsp90) is a molecular chaperone regulating the activity of diverse client proteins together with a plethora of different co-chaperones. Whether these functionally cooperate has remained enigmatic. We analyze all double mutants of 11 Saccharomyces cerevisiae Hsp90 co-chaperones in vivo concerning effects on cell physiology and the activation of specific client proteins.

Functional Characterisation of ClpP Mutations Conferring Resistance to Acyldepsipeptide Antibiotics in Firmicutes.

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes.

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search.

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer.

Genetic Code Expansion, Protein Expression, and Protein Functionalization in .

The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host . This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry.

The Heat Shock Response in Yeast Maintains Protein Homeostasis by Chaperoning and Replenishing Proteins.

Life is resilient because living systems are able to respond to elevated temperatures with an ancient gene expression program called the heat shock response (HSR). In yeast, the transcription of hundreds of genes is upregulated at stress temperatures. Besides stress protection conferred by chaperones, the function of the majority of the upregulated genes under stress has remained enigmatic.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and .

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of -methylcytosine (mC) in RNA- and single-stranded DNA oligonucleotides, demethylation of -methyladenosine (mA) in tRNA or formation of 5-formyl cytosine (fC) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria.

Neocarzilin A Is a Potent Inhibitor of Cancer Cell Motility Targeting VAT-1 Controlled Pathways.

The natural product neocarzilin A () was discovered decades ago, and despite its potent cytotoxic effects no mode of action studies have been performed up to date. Synthesis of neocarzilins A, B, and C and a stereoisomer of provided insights into structural preferences as well as access to probes for functional studies.  turned out to be the most active member and was not only effective against cell proliferation but also migration, a novel and so far overlooked activity.

Nucleotide excision repair of abasic DNA lesions.

Apurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER.

An Interspecies Analysis Reveals Molecular Construction Principles of Interleukin 27.

Interleukin 27 (IL-27) is a cytokine that regulates inflammatory responses. It is composed of an α subunit (IL-27α) and a β subunit (EBI3), which together form heterodimeric IL-27. Despite this general principle, IL-27 from different species shows distinct characteristics: Human IL-27α is not secreted autonomously while EBI3 is.

Publisher Correction: A network of chaperones prevents and detects failures in membrane protein lipid bilayer integration.

The original version of this Article contained errors in Fig. 1 and Supplementary Fig. 3.

A network of chaperones prevents and detects failures in membrane protein lipid bilayer integration.

A fundamental step in membrane protein biogenesis is their integration into the lipid bilayer with a defined orientation of each transmembrane segment. Despite this, it remains unclear how cells detect and handle failures in this process. Here we show that single point mutations in the membrane protein connexin 32 (Cx32), which cause Charcot-Marie-Tooth disease, can cause failures in membrane integration.

Ribosomal Peptides and Small Proteins on the Rise.

Genetically encoded and ribosomally synthesised peptides and small proteins act as important regulators in fundamental cellular processes, including gene expression, development, signalling and metabolism. Moreover, they also play a crucial role in eukaryotic and prokaryotic defence against microorganisms. Extremely diverse in size and structure, they are often subject to extensive post-translational modification.

Dissipative Self-Assembly of Photoluminescent Silicon Nanocrystals.

Solutions of silicon nanocrystals (SiNCs) are used in a diverse range of applications because of their tunable photoluminescence, biocompatibility, and the abundance of Si. In dissipative supramolecular materials, self-assembly of molecules or nanoparticles is driven by a chemical reaction network that irreversible consumes fuel. The properties of the emerging structures are controlled by the kinetics of the underlying chemical reaction network.

A Sulfoxide-Based Isobaric Labelling Reagent for Accurate Quantitative Mass Spectrometry.

Modern proteomics requires reagents for exact quantification of peptides in complex mixtures. Peptide labelling is most typically achieved with isobaric tags that consist of a balancer and a reporter part that separate in the gas phase. An ingenious distribution of stable isotopes provides multiple reagents with identical molecular weight but a different mass of the reporter groups, allowing relative quantification of multiple samples in one measurement.

Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter.

Enzymatic oxidation of 5-methylcytosine (5-mC) in the CpG dinucleotides to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) has central role in the process of active DNA demethylation and epigenetic reprogramming in mammals. However, it is not known whether the 5-mC oxidation products have autonomous epigenetic or regulatory functions in the genome. We used an artificial upstream promoter constituted of one cAMP response element (CRE) to measure the impact of 5-mC in a hemi-methylated CpG on the promoter activity and further explored the consequences of 5-hmC, 5-fC, and 5-caC in the same system.

A Chemical Disruptor of the ClpX Chaperone Complex Attenuates the Virulence of Multidrug-Resistant Staphylococcus aureus.

The Staphylococcus aureus ClpXP protease is an important regulator of cell homeostasis and virulence. We utilized a high-throughput screen against the ClpXP complex and identified a specific inhibitor of the ClpX chaperone that disrupts its oligomeric state. Synthesis of 34 derivatives revealed that the molecular scaffold is restrictive for diversification, with only minor changes tolerated.

Synthesis of RNA Containing 5-Hydroxymethyl-, 5-Formyl-, and 5-Carboxycytidine.

5-Hydroxymethyl-, 5-formyl-, and 5-carboxy-2'-deoxycytidine are new epigenetic bases (hmdC, fdC, cadC) that were recently discovered in the DNA of higher eukaryotes. The same bases (5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine; hmC, fC, and caC) have now also been detected in mammalian RNA with a high abundance in mRNA. While DNA phosphoramidites (PAs) that allow the synthesis of xdC-containing oligonucleotides for deeper biological studies are available, the corresponding silyl-protected RNA PAs for fC and caC have not yet been disclosed.

The Plasticity of the Hsp90 Co-chaperone System.

The Hsp90 system in the eukaryotic cytosol is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function. Although progress has been made regarding the underlying biochemical mechanisms, how co-chaperones influence Hsp90 client proteins in vivo has remained elusive. By investigating the effect of 12 Hsp90 co-chaperones on the activity of different client proteins in yeast, we find that deletion of co-chaperones can have a neutral or negative effect on client activity but can also lead to more active clients.