PARP1 proximity proteomics reveals interaction partners at stressed replication forks.

PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.

Proteomic analysis of tyrosine phosphorylation induced by exogenous expression of oncogenic kinase fusions identified in lung adenocarcinoma.

Kinase fusions are considered oncogenic drivers in numerous types of cancer. In lung adenocarcinoma 5-10% of patients harbor kinase fusions. The most frequently detected kinase fusion involves the Anaplastic Lymphoma Kinase (ALK) and Echinoderm Microtubule-associated protein-Like 4 (EML4). In addition, oncogenic kinase fusions involving the tyrosine kinases RET and ROS1 are found in smaller subsets of patients. In this study, we employed quantitative mass spectrometry-based phosphoproteomics to define the cellular tyrosine phosphorylation patterns induced by different oncogenic kinase fusions identified in patients with lung adenocarcinoma. We show that exogenous expression of the kinase fusions in HEK 293T cells leads to widespread tyrosine phosphorylation. Direct comparison of different kinase fusions demonstrates that the kinase part and not the fusion partner primarily defines the phosphorylation pattern. The tyrosine phosphorylation patterns differed between ALK, ROS1, and RET fusions, suggesting that oncogenic signaling induced by these kinases involves the modulation of different cellular processes.

Proteomic profiling of VCP substrates links VCP to K6-linked ubiquitylation and c-Myc function.

Valosin-containing protein (VCP) is an evolutionarily conserved ubiquitin-dependent ATPase that mediates the degradation of proteins through the ubiquitin-proteasome pathway. Despite the central role of VCP in the regulation of protein homeostasis, identity and nature of its cellular substrates remain poorly defined. Here, we combined chemical inhibition of VCP and quantitative ubiquitin remnant profiling to assess the effect of VCP inhibition on the ubiquitin-modified proteome and to probe the substrate spectrum of VCP in human cells. We demonstrate that inhibition of VCP perturbs cellular ubiquitylation and increases ubiquitylation of a different subset of proteins compared to proteasome inhibition. VCP inhibition globally upregulates K6-linked ubiquitylation that is dependent on the HECT-type ubiquitin E3 ligase HUWE1. We report ~450 putative VCP substrates, many of which function in nuclear processes, including gene expression, DNA repair and cell cycle. Moreover, we identify that VCP regulates the level and activity of the transcription factor c-Myc.

ATR inhibition rewires cellular signaling networks induced by replication stress.

The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling.

RAD54L2 counters TOP2-DNA adducts to promote genome stability.

The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

Development of a microwell adapted immunoblot system with recombinant antigens for distinguishing human herpesvirus (HHV)6A and HHV6B and detection of human cytomegalovirus.

BACKGROUND: The human cytomegalovirus (HCMV) and the human herpesvirus 6 (HHV6) are widely distributed in the human population. The variants A and B of HHV6 are closely related to each other and cannot be distinguished by common serological methods like enzyme-linked immunosorbent assay (ELISA) or immunofluorescence test (IFT). The aim of this study was to develop a microwell-adapted blot system for specificity detection of human cytomegalovirus and human herpesvirus 6A and 6B (HHV6A, HHV6B) that combines the advantages of ELISA (automation and multiplex detection) and immunoblotting (antigen-specific antibody detection with high specificity). METHODS: Ten HCMV, five HHV6A and five HHV6B antigens were expressed as fusion proteins and tested with sera of children (n=30), of healthy young adults (n=30) and of older adults (n=30) in a newly developed microblot system. RESULTS: Sensitivity and specificity of HCMV and HHV6 microblots were comparable to commercially available[ELISA, IFT and to line assay tests. The advantage of the HHV6 microblot is the possibility of distinguishing between HHV6A-monovalent sera, HHV6B-monovalent sera and HHV6A/B-polyvalent sera. Most sera of children younger than 2 years showed only HHV6B antigen positivity, while most sera of adults and children aged over 2 years reacted with HHV6A and B proteins, although predominance for HHV6B was observed. CONCLUSIONS: The authors were able to detect HCMV positive sera and to distinguish between HHV6A-monovalent sera, HHV6B-monovalent sera and HHVA/B-polyvalent sera with the new developed microblot system. Predominance of HHV6B was observed in sera of children and adults.

The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation.

BACKGROUND: Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control. RESULTS: Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1. CONCLUSIONS: We propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins as primary substrates and 14-3-3 as direct readers of p38-MK2-dependent phosphorylation induced by UV light. Mechanistically, we show that MK2 phosphorylates the RNA-binding subunit of the NELF complex NELFE on Serine 115. NELFE phosphorylation promotes the recruitment of 14-3-3 and rapid dissociation of the NELF complex from chromatin, which is accompanied by RNA polymerase II elongation.

Interaction profiling of RNA-binding ubiquitin ligases reveals a link between posttranscriptional regulation and the ubiquitin system.

RNA-binding ubiquitin ligases (RBULs) have the potential to link RNA-mediated mechanisms to protein ubiquitylation. Despite this, the cellular functions, substrates and interaction partners of most RBULs remain poorly characterized. Affinity purification (AP) combined with quantitative mass spectrometry (MS)-based proteomics is a powerful approach for analyzing protein functions. Mapping the physiological interaction partners of RNA-binding proteins has been hampered by their intrinsic properties, in particular the existence of low-complexity regions, which are prone to engage in non-physiological interactions. Here, we used an adapted AP approach to identify the interaction partners of human RBULs harboring different RNA-binding domains. To increase the likelihood of recovering physiological interactions, we combined control and bait-expressing cells prior to lysis. In this setup, only stable interactions that were originally present in the cell will be identified. We exploit gene function similarity between the bait proteins and their interactors to benchmark our approach in its ability to recover physiological interactions. We reveal that RBULs engage in stable interactions with RNA-binding proteins involved in different steps of RNA metabolism as well as with components of the ubiquitin conjugation machinery and ubiquitin-binding proteins. Our results thus demonstrate their capacity to link posttranscriptional regulation with the ubiquitin system.

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts.

The receptor components of the chloroplast protein import machinery, Toc34 and Toc159, are both encoded by small gene families in Arabidopsis thaliana. Recent results suggest that each member of these families preferentially interacts with different groups of precursor proteins. Here we address the question, whether multiple homologous Toc receptors are unique to Arabidopsis or whether they are a general phenomenon in plants. Indeed, in spinach we could identify at least two Toc34 proteins with different substrate specificities as demonstrated by competition and antibody inhibition experiments. In addition, an analysis of the available genomic data revealed the presence of at least two Toc34 homologs in six other plant species.

Studies on differential nuclear translocation mechanism and assembly of the three subunits of the Arabidopsis thaliana transcription factor NF-Y.

The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF-YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

Manual abilities of the elderly--handgrip strength, finger and thumb push strength and opening strength in age comparison.

The purpose of this paper is to display the static strength capacities of healthy adults in different age categories. A total of 279 healthy German adults at the ages of 20 to 29 years, 50 to 59 years and 60 to 69 years generated their maximum static handgrip, index finger and thumb push strength, as well as their maximum opening strength on a smooth jar lid of 85 mm diameter and on a knurled bottle lid of 31 mm with their right hand. The results show larger male strength than female strength. Significant age-induced differences appear primarily in opening strengths between the age groups 20 to 29 and 50 to 59 years in male subjects and in female opening strengths between the age groups 20 to 29 and 60 to 69 years as well as between the age groups 50 to 59 and 60 to 69 years. Of greatest interest is that elderly men show the largest opening strengths.

Body measurements and the variability of sitting postures at preschool age as preconditions for an optimal adjustment of chairs and tables.

Preschool age is a biological stage of intensive longitudinal growth with high plasticity of the growing body and of body postures. It is the period where children learn to persist in a sitting posture for a longer time and to use furniture like chairs or other body supporting systems. The growing body shows a special sensitivity for the manifestation of inappropriate postures. In this study the development of body measurements and sitting behaviour of preschool age children is investigated as a precondition for an optimal adjustment of seats and desks to the growing body. Accordingly to the instructions of Knussmann (1988) and Jiirgens (1988) 6 body measurements were taken from 122 German children aged 3 to 7 years from Potsdam, Province Brandenburg. Additionally, every child was videotaped for 10 minutes while crayoning in a sitting position of its own choice using a chair and a desk. To analyse the tapes, the software Noldus Observer was used and examined, picture by picture, to define the different types of sitting postures as well as the duration of persistence in a posture and the number of changes of postures. The used chairs and desks were also measured. Furthermore, the data of the furniture guideline DIN ISO 5970 (DIN, 1981), which regulates the dimensions of furniture for sitting in educational institutions, were compared with the results of the body measurements and with the dimensions of the furniture used by the children.