Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain [Protein Structure and Folding]

September 24th, 2015 by Selzer, L., Kant, R., Wang, J. C.-Y., Bothner, B., Zlotnick, A.

Hepatitis B Virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty HBV T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage compared to CTDs of Cp183-WT capsids. Cryo-EM studies of trypsin-digested capsids show that CTDs at fivefold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at fivefolds, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the HBV lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.
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Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle [Cell Biology]

September 24th, 2015 by

Small Ankyrin 1 (sAnk1) is a 17 kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum (nSR) in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in SR vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation (coIP) and an anisotropy-based FRET method (AFRET). Binding was reduced approximately two-fold by the replacement of all the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of SERCA1s apparent Ca2+ affinity, but that sAnk1s effect is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca2+ in the SR lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca2+ misregulation.
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The protein complex of neurodegeneration-related phosphoinositide phosphatase Sac3 and ArPIKfyve binds the Lewy-body-associated Synphilin-1 preventing its aggregation [Neurobiology]

September 24th, 2015 by

The 5-phosphoinositide phosphatase Sac3, whose loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders.
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Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex [Cell Biology]

September 24th, 2015 by

The main target of cAMP is protein kinase A (PKA), the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: Acrodysostosis and Carney complex. In addition to the initial recurrent mutation (Arg368X) of PRKAR1A gene, several missense and nonsense mutations have been recently observed in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP binding domains of the protein and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-PIP-cAMP) or domain B (8-AHA-cAMP) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis, but the existence of several independent mechanisms leading to constitutive PKA activation in Carney complex.
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Promoter escape with bacterial two-component sigma factor suggests retention of sigma region two in the elongation complex [Gene Regulation]

September 23rd, 2015 by Sengupta, S., Prajapati, R. K., Mukhopadhyay, J.

The transition from the formation of RNAP-promoter open complex step to productive elongation complex step involves promoter escape of RNAP. From the structure of RNAP, a promoter escape model has been proposed which suggests that the interactions between σR4 and RNAP and σR4 and DNA are destabilized upon transition to elongation. This accounts for reduced affinity of σ to RNAP and stochastic release of σ. However, as the loss of interaction of σR4 with RNAP results in the release of intact σ, assessing this interaction remains challenging to be experimentally verified. Here we study the promoter escape model using a two- component σ factor YvrI and YvrHa from Bacillus subtilis that independently contribute to the functions of σR4 and σR2 in a RNAP-promoter complex. Our results show that YvrI, that mimics σR4, is released gradually as transcription elongation proceeds whereas YvrHa that mimics σR2 is retained throughout the elongation complexes. Thus our result validates the proposed model for promoter escape and also suggests that promoter escape involves little or no change in the interaction of σR2 with RNAP.

Ablation of ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis [Molecular Bases of Disease]

September 23rd, 2015 by Chen, L., Hambright, W. S., Na, R., Ran, Q.

Glutathione peroxidase 4(Gpx4), an antioxidant defense enzyme in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that Gpx4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis, and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in spinal cord, but had no overt neuron degeneration in cerebral cortex. Consistent with Gpx4′s role as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplement of vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. And lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by Gpx4 is essential for motor neuron health and survival in vivo.
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Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic {alpha}{beta}T cell Receptor Chains [Protein Structure and Folding]

September 23rd, 2015 by Feige, M. J., Behnke, J., Mittag, T., Hendershot, L. M.

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control (ERQC) decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here, we used the clonotypic αβ chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ERQC with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αβTCR assembly control in the cell. We found that folding of the TCR α-chain constant domain Cα is dependent on αβ-heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/β assembly process that allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and thus reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α- and β-chain combinations that will comprise the T cell repertoire.
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Cystathionine {beta}-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria [Signal Transduction]

September 23rd, 2015 by

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, Family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP, ADP) or activation (ATP). Here we show that linear P1,Pn-diadenosine 5′-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi and Clostridium perfringens, and increase their activity up to 30-, 5- and 7-fold, respectively. Ap4A, Ap5A and Ap6A bound non-cooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were approximately 10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A, but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.
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Antigen-B Cell Receptor Complexes Associate with Intracellular MHC Class II Molecules [Cell Biology]

September 23rd, 2015 by Barroso, M., Tucker, H., Drake, L., Nichol, K., Drake, J. R.

Antigen processing and MHC class II-restricted antigen presentation by antigen presenting cells (APC) such as dendritic cells and B cells allows for activation of naive CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted APC in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging / FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, previously shown to be critical for CD4 T cell activation, is selectively incorporated into these complexes and selectively loaded with peptide derived from BCR-internalized cognate antigen. These results demonstrate that in B cells internalized Ag-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms that B cells use to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.

Insights into the determination of the templating nucleotide at the initiation of ϕ29 DNA replication [Enzymology]

September 23rd, 2015 by

Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognises the replication origins, placed at both 5' ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3' end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser232 is located, indicate that the aromatic residue Phe230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerisation active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe230 in limiting the internalisation of the template strand in the polymerisation active site is discussed.