Structural basis and regulation of the reductive stress response.

Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis.

Reengineering a tRNA Methyltransferase To Covalently Capture New RNA Substrates.

Covalent RNA modifications can alter the function and metabolism of RNA transcripts. Altering the RNA substrate specificities of the enzymes that install these modifications can provide powerful tools to study and manipulate RNA. To develop new tools and probe the plasticity of the substrate specificity of one of these enzymes, we examined the engineerability of the uridine-54 tRNA methyltransferase, TrmA. Starting from a mutant that remains covalently bound to its substrate RNA (E358Q, TrmA*), we were able to use both rational design and a high-throughput sequencing assay to examine the RNA substrates of TrmA*. Although rational engineering substantially changed TrmA* specificity, the rationally designed substrate mutants we developed still retained activity with the wild-type protein. Using high-throughput substrate screening of additional TrmA* mutants, we identified a triple mutant of the substrate RNA (C56A;A58G;C60U) that is efficiently trapped by a TrmA* double mutant (E49R;R51E) but not by the wild-type TrmA*. This work establishes a foundation for using protein engineering to reconfigure substrate specificities of RNA-modifying enzymes and covalently trap RNAs with engineered proteins.

Requirements for efficient endosomal escape by designed mini-proteins.

ZF5.3 is a compact, rationally designed mini-protein that escapes efficiently from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells and guides multiple classes of proteins into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher. But other than the requirement for unfoldable cargo and an intact HOPS complex, there is little known about how ZF5.3 traverses the limiting endocytic membrane. Here we delineate the attributes of ZF5.3 that enable efficient endosomal escape. We confirm that ZF5.3 is stable at pH values between 5.5 and 7.5, with no evidence of unfolding even at temperatures as high as 95 °C. The high-resolution NMR structure of ZF5.3 at pH 5.5, also reported here, shows a canonical p zinc-finger fold with the penta-arg motif integrated seamlessly into the C-terminal α-helix. At lower pH, ZF5.3 unfolds cooperatively as judged by both circular dichroism and high-resolution NMR. Unfolding occurs upon protonation of a single Zn(II)-binding His side chain whose pKa corresponds almost exactly to that of the late endosomal lumen. pH-induced unfolding is essential for endosomal escape, as a ZF5.3 analog that remains folded at pH 4.5 fails to efficiently reach the cytosol, despite high overall uptake. Finally, using reconstituted liposomes, we identify a high-affinity interaction of ZF5.3 with a specific lipid-BMP-that is selectively enriched in the inner leaflet of late endosomal membranes. This interaction is 10-fold stronger at low pH than neutral pH, providing a molecular picture for why escape occurs preferentially and in a HOPS-dependent manner from late endosomal compartments. The requirements for programmed endosomal escape identified here should aid and inform the design of proteins, peptidomimetics, and other macromolecules that reach cytosolic or nuclear targets intact and at therapeutically relevant concentrations.

HOPS-Dependent Endosomal Escape Demands Protein Unfolding.

The inefficient translocation of proteins across biological membranes limits their application as potential therapeutics and research tools. In many cases, the translocation of a protein involves two discrete steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules can achieve high protein uptake, but few are capable of efficient endosomal escape. One exception to this rule is ZF5.3, a mini-protein that exploits elements of the natural endosomal maturation machinery to translocate across endosomal membranes. Although some ZF5.3-protein conjugates are delivered efficiently to the cytosol or nucleus, overall delivery efficiency varies widely for different cargoes with no obvious design rules. Here we show that delivery efficiency depends on the ability of the cargo to unfold. Using fluorescence correlation spectroscopy, a single-molecule technique that precisely measures intracytosolic protein concentration, we show that regardless of size and pI, low-Tm cargoes of ZF5.3 (including intrinsically disordered domains) bias endosomal escape toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal, even if the Tm is high. These findings imply a novel pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.

Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2.

The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF-tMeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13-71, 313-484), binds DNA in a methylation-dependent manner in vitro, and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF-tMeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.

Design rules for efficient endosomal escape.

The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a single-molecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.

Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. METHODS: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). RESULTS: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. CONCLUSION: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.