Proteomic identification of proliferation and progression markers in human polycythemia vera stem and progenitor cells.

Polycythemia vera (PV) is a stem cell disorder characterized by hyperproliferation of the myeloid lineages and the presence of an activating JAK2 mutation. To elucidate mechanisms controlling PV stem and progenitor cell biology, we applied a recently developed highly sensitive data-independent acquisition mass spectrometry workflow to purified hematopoietic stem and progenitor cell (HSPC) subpopulations of patients with chronic and progressed PV. We integrated proteomic data with genomic, transcriptomic, flow cytometry, and in vitro colony formation data. Comparative analyses revealed added information gained by proteomic compared with transcriptomic data in 30% of proteins with changed expression in PV patients. Upregulated biological pathways in hematopoietic stem and multipotent progenitor cells (HSC/MPPs) of PV included mammalian target of rapamycin (MTOR), STAT, and interferon signaling. We further identified a prominent reduction of clusterin (CLU) protein expression and a corresponding activation of nuclear factor-κB (NF-κB) signaling in HSC/MPPs of untreated PV patients compared with controls. Reversing the reduction of CLU and inhibiting NF-κB signaling decreased proliferation and differentiation of PV HSC/MPPs in vitro. Upon progression of PV, we identified upregulation of LGALS9 and SOCS2 protein expression in HSC/MPPs. Treatment of patients with hydroxyurea normalized the expression of CLU and NF-κB2 but not of LGALS9 and SOCS2. These findings expand the current understanding of the molecular pathophysiology underlying PV and provide new potential targets (CLU and NF-κB) for antiproliferative therapy in patients with PV.

The conscientiousness-health link in depression: Results from a path analysis.

BACKGROUND: This was the first study aiming to develop a theory-based model to analyze the relation between conscientiousness and impaired physical health as well as between conscientiousness and subjective health-related well-being within a large clinical sample with depressive symptomatology. In addition, a mediating effect of health risk behaviors regarding the association between conscientiousness and objectively impaired physical health as well as subjective health-related well-being were exploratory examined. METHODS: Individuals with depressive symptoms (n = 943) with a mean age of 52 years (ranging between 20 and 78 years) undergoing intensive psychiatric rehabilitation treatment were investigated with the Big Five Inventory-10 as well as several self-report health questionnaires (Three-Factor Eating Questionnaire, Food-Craving Inventory and lifestyle questions assessing physical inactivity and alcohol consumption). Health-related well-being was measured using the World Health Organisation Quality of Life Assessment and the construct of impaired physical health comprised anthropometric measurements (Body Mass Index, Waist-to-Height-Ratio), blood lipids, and impaired physical performance capacity on the bicycle-ergometric test. RESULTS: Structural path analyses revealed that unhealthy eating habits and physical inactivity partially mediated the negative relation between conscientiousness and impaired physical health as well as the positive relation between conscientiousness and health-related well-being. LIMITATIONS: Possible limitations include cross-sectional study design, missing data, assessment of conscientiousness on a global level and self-report assessment of health risk behaviors. CONCLUSIONS: The findings highlight the importance concerning the extended inclusion of personality aspects in the treatment of depression in order to improve health.

The protein landscape of chronic lymphocytic leukemia.

Many functional consequences of mutations on tumor phenotypes in chronic lymphocytic leukemia (CLL) are unknown. This may be in part due to a scarcity of information on the proteome of CLL. We profiled the proteome of 117 CLL patient samples with data-independent acquisition mass spectrometry and integrated the results with genomic, transcriptomic, ex vivo drug response, and clinical outcome data. We found trisomy 12, IGHV mutational status, mutated SF3B1, trisomy 19, del(17)(p13), del(11)(q22.3), mutated DDX3X and MED12 to influence protein expression (false discovery rate [FDR] = 5%). Trisomy 12 and IGHV status were the major determinants of protein expression variation in CLL as shown by principal-component analysis (1055 and 542 differentially expressed proteins, FDR = 5%). Gene set enrichment analyses of CLL with trisomy 12 implicated B-cell receptor (BCR)/phosphatidylinositol 3-kinase (PI3K)/AKT signaling as a tumor driver. These findings were supported by analyses of protein abundance buffering and protein complex formation, which identified limited protein abundance buffering and an upregulated protein complex involved in BCR, AKT, MAPK, and PI3K signaling in trisomy 12 CLL. A survey of proteins associated with trisomy 12/IGHV-independent drug response linked STAT2 protein expression with response to kinase inhibitors, including Bruton tyrosine kinase and mitogen-activated protein kinase kinase (MEK) inhibitors. STAT2 was upregulated in unmutated IGHV CLL and trisomy 12 CLL and required for chemokine/cytokine signaling (interferon response). This study highlights the importance of protein abundance data as a nonredundant layer of information in tumor biology and provides a protein expression reference map for CLL.

The iron-sulphur cluster in human DNA2 is required for all biochemical activities of DNA2.

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2's nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding.

The iron-sulfur helicase DDX11 promotes the generation of single-stranded DNA for CHK1 activation.

The iron-sulfur (FeS) cluster helicase DDX11 is associated with a human disorder termed Warsaw Breakage Syndrome. Interestingly, one disease-associated mutation affects the highly conserved arginine-263 in the FeS cluster-binding motif. Here, we demonstrate that the FeS cluster in DDX11 is required for DNA binding, ATP hydrolysis, and DNA helicase activity, and that arginine-263 affects FeS cluster binding, most likely because of its positive charge. We further show that DDX11 interacts with the replication factors DNA polymerase delta and WDHD1. In vitro, DDX11 can remove DNA obstacles ahead of Pol δ in an ATPase- and FeS domain-dependent manner, and hence generate single-stranded DNA. Accordingly, depletion of DDX11 causes reduced levels of single-stranded DNA, a reduction of chromatin-bound replication protein A, and impaired CHK1 phosphorylation at serine-345. Taken together, we propose that DDX11 plays a role in dismantling secondary structures during DNA replication, thereby promoting CHK1 activation.

WRNIP1 Protects Reversed DNA Replication Forks from SLX4-Dependent Nucleolytic Cleavage.

During DNA replication stress, stalled replication forks need to be stabilized to prevent fork collapse and genome instability. The AAA + ATPase WRNIP1 (Werner Helicase Interacting Protein 1) has been implicated in the protection of stalled replication forks from nucleolytic degradation, but the underlying molecular mechanism has remained unclear. Here we show that WRNIP1 exerts its protective function downstream of fork reversal. Unexpectedly though, WRNIP1 is not part of the well-studied BRCA2-dependent branch of fork protection but seems to protect the junction point of reversed replication forks from SLX4-mediated endonucleolytic degradation, possibly by directly binding to reversed replication forks. This function is specific to the shorter, less abundant, and less conserved variant of WRNIP1. Overall, our data suggest that in the absence of BRCA2 and WRNIP1 different DNA substrates are generated at reversed forks but that nascent strand degradation in both cases depends on the activity of exonucleases and structure-specific endonucleases.

Human DNA polymerase delta requires an iron-sulfur cluster for high-fidelity DNA synthesis.

Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron-sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data hence suggest that the FeS cluster in human Pol δ is an important co-factor that despite its C-terminal location has an impact on both DNA polymerase and exonuclease activities, and can influence the fidelity of DNA synthesis.