Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

Decoding Cancer Variants of Unknown Significance for Helicase-Nuclease-RPA Complexes Orchestrating DNA Repair During Transcription and Replication.

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

GRB2 enforces homology-directed repair initiation by MRE11.

DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

A collagen glucosyltransferase drives lung adenocarcinoma progression in mice.

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning.

The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates Pol II to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in vitro in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning.

Envisioning how the prototypic molecular machine TFIIH functions in transcription initiation and DNA repair.

Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.

Effects of weight reduction on the breast cancer-related lymphedema: A systematic review and meta-analysis.

BACKGROUND: Obesity has long been considered a risk factor for breast cancer-related lymphedema (BCRL), but the benefits of weight reduction in managing BCRL have not been clearly established. OBJECTIVE: To evaluate the beneficial effects of weight loss interventions (WLIs) on the reduction and prevention of BCRL. METHODS: We conducted a systematic review and meta-analysis by searching the PubMed, Scopus, and Embase databases from their earliest record to October 1st, 2019. We included randomized and non-randomized controlled trials involving adult patients with a history of breast cancer, that compared WLI groups with no-WLI groups, and provided quantitative measurements of lymphedema. RESULTS: Initial literature search yielded 461 nonduplicate records. After exclusion based on title, abstract, and full-text review, four randomized controlled trials involving 460 participants were included for quantitative analysis. Our meta-analysis revealed a significant between-group mean difference (MD) regarding the volume of affected arm (MD = 244.7 mL, 95% confidence interval [CI]: 145.3-344.0) and volume of unaffected arm (MD = 234.5 mL, 95% CI: 146.9-322.1). However, a nonsignificant between-group MD of -0.07% (95% CI: 1.22-1.08) was observed regarding the interlimb volume difference at the end of the WLIs. CONCLUSIONS: In patients with BCRL, WLIs are associated with decreased volume of the affected and unaffected arms but not with decreased severity of BCRL measured by interlimb difference in arm volume.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation.

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

Author Correction: Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding.

In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to also include support from the National Institutes of Health grant T32GM008280 to Sarah Alvarado.

Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding.

Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded ß-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer.

The ATPase Motor Turns for Type IV Pilus Assembly.

In this issue of Structure, Mancl et al. (2016) elucidate the crystal structure of the PilB ATPase domain in complex with ATPγS and unveil how ATP binding and hydrolysis coordinates conformational change. Their results reveal a distinct symmetric rotary mechanism for ATP hydrolysis to power bacterial pilus assembly.

Emerging critical roles of Fe-S clusters in DNA replication and repair.

Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life - the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

Probing DNA by 2-OG-dependent dioxygenase.

TET-mediated 5-methyl cytosine (5mC) oxidation acts in epigenetic regulation, stem cell development, and cancer. Hu et al. now determine the crystal structure of the TET2 catalytic domain bound to DNA, shedding light on 5mC-DNA substrate recognition and the catalytic mechanism of 5mC oxidation.

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals.

DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging.

Friedreich's ataxia variants I154F and W155R diminish frataxin-based activation of the iron-sulfur cluster assembly complex.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ∼15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ∼ I154F > W155F > W155A ∼ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.

Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.

Cellular depletion of the human protein frataxin is correlated with the neurodegenerative disease Friedreich's ataxia and results in the inactivation of Fe-S cluster proteins. Most researchers agree that frataxin functions in the biogenesis of Fe-S clusters, but its precise role in this process is unclear. Here we provide in vitro evidence that human frataxin binds to a Nfs1, Isd11, and Isu2 complex to generate the four-component core machinery for Fe-S cluster biosynthesis. Frataxin binding dramatically changes the K(M) for cysteine from 0.59 to 0.011 mM and the catalytic efficiency (k(cat)/K(M)) of the cysteine desulfurase from 25 to 7900 M⁻¹s⁻¹. Oxidizing conditions diminish the levels of both complex formation and frataxin-based activation, whereas ferrous iron further stimulates cysteine desulfurase activity. Together, these results indicate human frataxin functions with Fe(2+) as an allosteric activator that triggers sulfur delivery and Fe-S cluster assembly. We propose a model in which cellular frataxin levels regulate human Fe-S cluster biosynthesis that has implications for mitochondrial dysfunction, oxidative stress response, and both neurodegenerative and cardiovascular disease.