Cell polarity kinase MST4 cooperates with cAMP-dependent kinase to orchestrate histamine-stimulated acid secretion in gastric parietal cells [Cell Biology]

September 24th, 2015 by

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which this signaling pathway operates in gastric acid secretion. Here we show PKA cooperates with MST4 to orchestrate histamine-elicited acid secretion by phosphorylating ezrin at Ser66 and Thr567, respectively. Histamine stimulation activates PKA which phosphorylates MST4 at Thr178 and then promotes MST4 kinase activity. Interestingly, activated MST4 then phosphorylates ezrin pre-phosphorylated by PKA. Importantly, MST4 is important for acid secretion in parietal cells because either suppression of MST4 or overexpression of non-phosphorylatable MST4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, overexpressing MST4 phosphorylation-deficient ezrin results in an inhibition of gastric acid secretion. Taken together, these results define a novel molecular mechanism linking PKA-MST4-ezrin signaling cascade to polarized epithelial secretion in gastric parietal cells.
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Structural Studies of IRF4 Reveal a Flexible Autoinhibitory Region and a Compact Linker Domain [Gene Regulation]

September 24th, 2015 by Remesh, S. G., Santosh, V., Escalante, C. R.

IRF4 is a unique member of the IRF family playing critical regulatory roles in immune cell development, regulation of obesity-induced inflammation and control of thermogenic gene expression. The ability of IRF4 to control diverse transcriptional programs arises from its proficiency to interact with numerous transcriptional partners. In this study we present the structural characterization of full length IRF4. Using a combination of X-ray and SAXS studies, we reveal unique features of the Interferon Activation Domain (IAD) including a set of β-sheets and loops that serve as the binding site for PU.1 and also show that unlike other IRF members, IRF4 has a flexible autoinhibitory region. In addition, we have determined the SAXS solution structure of full length IRF4 that together with circular dichroism studies suggest that the linker region is not extended but folds into a domain structure.

Assembling a correctly folded and functional heptahelical membrane protein by protein trans-splicing [Membrane Biology]

September 24th, 2015 by

Protein trans-splicing using split inteins is well established as useful tool for protein engineering. Here we show for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A-B and C-D-E-F-G via a splicing site located in the BC loop. The procedure presented here is based on dual expression and ligation in vivo. Global fold, stability and photodynamic were analyzed in detergent by CD-, stationary as well as time-resolved optical spectroscopy. Its fold within lipid bilayers has been probed by high field and DNP-enhanced solid-state NMR utilizing a 13Clabeled retinal co-factor and extensively 13C-15N labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins, for elucidating intramolecular interactions and they offer the possibility towards developing novel labeling schemes for spectroscopic applications.

Genomic survey and biochemical analysis of recombinant candidate cyanobacteriochromes reveals enrichment for near UV/Violet sensors in the halotolerant and alkaliphilic cyanobacterium Microcoleus IPPAS B353 [Genomics and Proteomics]

September 24th, 2015 by

Cyanobacteriochromes (CBCRs), which are exclusive to and widespread among cyanobacteria, are photoproteins that sense the entire range of near-UV and visible light. CBCRs are related to the red/far-red phytochromes that utilize linear tetrapyrrole (bilin) chromophores. Best characterized from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the multicellular heterocyst forming filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120, CBCRs have been poorly investigated in mat-forming, nonheterocystous cyanobacteria. In this study, we sequenced the genome of one of such species, Microcoleus IPPAS B353 (Microcoleus B353), and identified two phytochromes and seven CBCRs with one or more bilin-binding cGMP-specific phosphodiesterase, Adenylyl cyclase and FhlA (GAF) domains. Biochemical and spectroscopic measurements of 23 purified GAF proteins from phycocyanobilin (PCB) producing recombinant E. coli indicated that 13 of these proteins formed near-UV and visible light-absorbing covalent adducts: ten GAFs contained PCB chromophores whereas three contained the PCB isomer, phycoviolobilin (PVB). Furthermore, the complement of Microcoleus B353 CBCRs is enriched in near-UV and violet sensors, but lacks red/green and green/red CBCRs that are widely distributed in other cyanobacteria. We hypothesize that enrichment in short wavelength-absorbing CBCRs is critical for acclimation to high-light environments where this organism is found.
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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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