Uncovering the early assembly mechanism for amyloidogenic {beta}2-microglobulin using cross-linking and native mass spectrometry [Computational Biology]

December 11th, 2015 by Hall, Z., Schmidt, C., Politis, A.

β2-microglobulin (β2m), a key component of the major histocompatibility class I complex , can aggregate into fibrils with severe clinical consequences. As such, investigating structural aspects of the formation of oligomeric intermediates of β2m and their subsequent progression towards fibrillar aggregates is of great importance. However β2m aggregates are challenging targets in structural biology, primarily due to their inherent transient and heterogeneous nature. Here we study the oligomeric distributions and structures of the early intermediates of amyloidogenic β2m and its truncated variant ΔN6-β2m. We established compact oligomers for both variants by integrating advanced mass spectrometric techniques with available electron microscopy maps and atomic-level structures from NMR spectroscopy and X-ray crystallography. Our results revealed a stepwise assembly mechanism by monomer addition and domain swapping, for the oligomeric species of (ΔN6)-β2m. The observed structural similarity and common oligomerization pathway between the two variants is likely to enable ΔN6-β2m to cross-seed β2m fibrillation and allow the formation of mixed fibrils. We further determined the key subunit interactions in ΔN6-β2m tetramer, revealing the importance of a domain-swapped hinge region for formation of higher-order oligomers. Overall, we deliver new mechanistic insights into β2m aggregation paving the way for future studies on the mechanisms and cause of amyloid fibrillation.
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Wilms tumor suppressor, WT1, cooperates with microRNA-26a and microRNA-101 to suppress translation of the Polycomb protein, EZH2, in mesenchymal stem cells [RNA]

December 10th, 2015 by

Hereditary forms of Wilms arise from developmentally arrested clones of renal progenitor cells with biallelic mutations of WT1; recently, it has been found that Wilms tumors may also be associated with biallelic mutations in DICER1 or DROSHA, crucial for miRNA biogenesis. We have previously shown that a critical role for WT1 during normal nephrogenesis is to suppress transcription of the Polycomb group protein, EZH2, thereby de-repressing genes in the differentiation cascade. Here we show that WT1 also suppresses translation of EZH2. All major WT1 isoforms induce an array of miRNAs, which target the 3[prime] UTR of EZH2 and other Polycomb associated transcripts. We show that the WT1(+KTS) isoform binds to the 5[prime] UTR of EZH2 and interacts directly with the miRNA-containing RISC to enhance post-transcriptional inhibition. These observations suggest a novel mechanism through which WT1 regulates the transition from resting stem cell to activated progenitor cell during nephrogenesis. Our findings also offer a plausible explanation for the fact that Wilms tumors can arise either from loss of WT1 or loss of miRNA processing enzymes.
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Molecular Determinants of Kv1.3 Potassium Channels-Induced Proliferation [Signal Transduction]

December 10th, 2015 by

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system, Kv1.5 overexpression decreases proliferation while Kv1.3 expression increases it independently of K+ fluxes. To identify Kv1.3 domains involved in proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C-terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Y447 and S459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Y447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C-terminus. For one of these sites (Y447) we demonstrated the contribution of MEK/ERK dependent phosphorylation, which is regulated by voltage-induced conformational changes.

Lipid-anchored synaptobrevin provides little or no support for exocytosis or liposome fusion [Membrane Biology]

December 8th, 2015 by

SNARE proteins catalyze many forms of biological membrane fusion including Ca2+-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear and prior studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it bears on the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems, Ca2+ triggered dense-core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca2+-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α (CSPα), which harbors many lipid acylation sites, provided support for fusion, but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-CSPα may reflect a slower alternate mode of SNARE-mediated membrane fusion.

Characterization of a linked Jumonji domain of the KDM5/JARID1 family of histone H3 lysine 4 demethylases [Gene Regulation]

December 8th, 2015 by

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and di-methylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems support a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally-characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 sub-families, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared to that of KDM5B at 1 mM α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was approximately 8-fold more potent against KDM5B than KDM5C. Interestingly, the relative selectivity of JIB-04 towards KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C- terminal helical Zn-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.

Interferon {gamma} (IFN{gamma})-Signaling via mTORC2 and Regulatory Effects in the Generation of Type II IFN Biological Responses [Signal Transduction]

December 8th, 2015 by

We provide evidence for a unique pathway engaged by the Type II IFN-receptor, involving mTORC2/AKT-mediated downstream regula-tion of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of Type II interferon stimulated genes (ISGs). Our studies establish that Rictor is essential for the generation of Type II IFN-dependent antiviral and antiproliferative responses and controls the generation of Type II IFN-suppressive effects on normal and malignant hematopoiesis. Altogether, our findings establish a central role for mTORC2 in IFNγ-signaling and Type II IFN responses.
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Molecular Basis of Filtering Carbapenems by Porins from {beta}-Lactam-resistant Clinical Strains of Escherichia coli [Molecular Bases of Disease]

December 8th, 2015 by

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28 and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology, differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavourable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.
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Mechanism of folding and activation of subtilisin kexin isozyme-1(SKI-1)/site-1 protease (S1P) [Protein Structure and Folding]

December 8th, 2015 by

The proprotein convertase subtilisin kexin isozyme-1(SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multi-step process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure, but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition possibly allowing more subtle fine-tuning of the maturation process.

Acetoacetate accelerates muscle regeneration and ameliorates muscular dystrophy in mice [Cell Biology]

December 8th, 2015 by

Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we uncovered a novel function of AA in promoting muscle cell proliferation. Notably, the functional role of AA in regulating muscle cell function is further evidenced by its capability to accelerates muscle regeneration in normal mice and ameliorates muscular dystrophy in mdx mice. Mechanistically, our data from multi-parameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. Finally, we show that AA exerts its function through activation of the Mek1-Erk1/2-cyclin D1 pathway, revealing a novel mechanism in which AA serves as a signaling metabolite in mediating muscle cell function. Our findings highlight profound functions of a small metabolite as signaling molecule in mammalian cells.

Regulation of the Water Channel Aquaporin-2 via 14-3-3 Theta ({theta}) and Zeta ({zeta}) [Membrane Biology]

December 8th, 2015 by

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of sigma (σ), all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3 beta (β) and zeta (ζ), whereas levels of 14-3-3 eta (η) and theta (θ) were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2:14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3 ζ and θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2:14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl-terminus, with ser256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3 ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life and reduced AQP2 levels. In contrast, knockdown of 14-3-3 θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3 θ and ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation and degradation.