NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.

Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4's AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4's β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates.

A goldilocks computational protocol for inhibitor discovery targeting DNA damage responses including replication-repair functions.

While many researchers can design knockdown and knockout methodologies to remove a gene product, this is mainly untrue for new chemical inhibitor designs that empower multifunctional DNA Damage Response (DDR) networks. Here, we present a robust Goldilocks (GL) computational discovery protocol to efficiently innovate inhibitor tools and preclinical drug candidates for cellular and structural biologists without requiring extensive virtual screen (VS) and chemical synthesis expertise. By computationally targeting DDR replication and repair proteins, we exemplify the identification of DDR target sites and compounds to probe cancer biology.

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons.

Unlabelled: Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntington's disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain.

Enriched G4 forming repeats in the human genome are associated with robust well-coordinated transcription and reduced cancer transcriptome variation.

Non-B DNA G-quadruplex (G4) structures with guanine (G) runs of 2 to 4 repeats can trigger opposing experimental transcriptional impacts. Here, we used bioinformatic algorithms to comprehensively assess correlations of steady-state RNA transcript levels with all putative G4 sequence (pG4) locations genome-wide in three mammalian genomes and in normal and tumor human tissues. The human pG4-containing gene set displays higher expression levels than the set without pG4, supporting and extending some prior observations.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TAR DNA-binding protein 43 (TDP43) is increasingly recognized for its involvement in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 proteinopathy, characterized by dysregulated nuclear export and cytoplasmic aggregation, is present in most ALS/FTD cases and is associated with a loss of nuclear function and genomic instability in neurons. Building on prior evidence linking TDP43 pathology to DNA double-strand breaks (DSBs), this study identifies a novel regulatory role for TDP43 in the DNA mismatch repair (MMR) pathway.

ASCC1 structures and bioinformatics reveal a novel helix-clasp-helix RNA-binding motif linked to a two-histidine phosphodiesterase.

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2).

A prismatic view of the epigenetic-metabolic regulatory axis in breast cancer therapy resistance.

Epigenetic regulation established during development to maintain patterns of transcriptional expression and silencing for metabolism and other fundamental cell processes can be reprogrammed in cancer, providing a molecular mechanism for persistent alterations in phenotype. Metabolic deregulation and reprogramming are thus an emerging hallmark of cancer with opportunities for molecular classification as a critical preliminary step for precision therapeutic intervention. Yet, acquisition of therapy resistance against most conventional treatment regimens coupled with tumor relapse, continue to pose unsolved problems for precision healthcare, as exemplified in breast cancer where existing data informs both cancer genotype and phenotype.

Chemical screening by time-resolved X-ray scattering to discover allosteric probes.

Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF).

Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells.

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs.

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 Kras-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.

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology.

We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy.

The NS1 protein of influenza B virus binds 5'-triphosphorylated dsRNA to suppress RIG-I activation and the host antiviral response.

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non-structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5'triphosphorylated double-stranded RNA (5'-ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response.

Dynamics of the DYNLL1-MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs.

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer.

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition.

A functional link between lariat debranching enzyme and the intron-binding complex is defective in non-photosensitive trichothiodystrophy.

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC).

Dynamic conformational switching underlies TFIIH function in transcription and DNA repair and impacts genetic diseases.

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD.

Dynamics of the DYNLL1/MRE11 complex regulates DNA end resection and recruitment of the Shieldin complex to DSBs.

Extent and efficacy of DNA end resection at DNA double strand break (DSB)s determines the choice of repair pathway. Here we describe how the 53BP1 associated protein DYNLL1 works in tandem with Shieldin and the CST complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1 where it limits end resection by binding and disrupting the MRE11 dimer.

Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish.

Background: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network.

Epigenetic-Metabolic Interplay in the DNA Damage Response and Therapeutic Resistance of Breast Cancer.

Therapy resistance is imposing a daunting challenge on effective clinical management of breast cancer. Although the development of resistance to drugs is multifaceted, reprogramming of energy metabolism pathways is emerging as a central but heterogenous regulator of this therapeutic challenge. Metabolic heterogeneity in cancer cells is intricately associated with alterations of different signaling networks and activation of DNA damage response pathways.