scholarly journals The structure of the RCAN1:CN complex explains the inhibition of and substrate recruitment by calcineurin

2020 ◽  
Vol 6 (27) ◽  
pp. eaba3681
Author(s):  
Yang Li ◽  
Sarah R. Sheftic ◽  
Simina Grigoriu ◽  
Charles D. Schwieters ◽  
Rebecca Page ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Active Site ◽  
Neurological Disorders ◽  
Critical Factor ◽  
Endogenous Inhibitor ◽  
Weak Inhibitor ◽  
Road Map ◽  
Biochemical Studies ◽  
Specific Manner ◽  
Multiple Routes

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the Ser/Thr phosphatase calcineurin (CN). It has been shown that excessive inhibition of CN is a critical factor for Down syndrome and Alzheimer’s disease. Here, we determined RCAN1’s mode of action. Using a combination of structural, biophysical, and biochemical studies, we show that RCAN1 inhibits CN via multiple routes: first, by blocking essential substrate recruitment sites and, second, by blocking the CN active site using two distinct mechanisms. We also show that phosphorylation either inhibits RCAN1-CN assembly or converts RCAN1 into a weak inhibitor, which can be reversed by CN via dephosphorylation. This highlights the interplay between posttranslational modifications in regulating CN activity. Last, this work advances our understanding of how active site inhibition of CN can be achieved in a highly specific manner. Together, these data provide the necessary road map for targeting multiple neurological disorders.

Sirtuin-dependent reversible lysine acetylation controls the activity of acetyl-Coenzyme A synthetase in Campylobacter jejuni

2021 ◽  
Author(s):  
Victoria L. Jeter ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Campylobacter Jejuni ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Protein Function ◽  
Metabolic Enzyme ◽  
Lysine Acetylation ◽  
Class Iii ◽  
Acetyl Coa

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group ( N ε ) of an active-site lysine of the AMP-forming acetyl-CoA synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, the alluded active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N -acetyltransferases (GNATs). Acs activity is lost as a result of acetylation, and restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c , cj1715, and cj1050c loci, namely the AMP-forming acetate:CoA ligase ( Cj Acs), a type IV GCN5-type lysine acetyltransferase (GNAT, hereafter Cj LatA), and a NAD + -dependent (class III) sirtuin deacylase ( Cj CobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of Cj Acs. IMPORTANCE This work is important because it provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase ( Cj Acs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of Cj Acs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

scholarly journals Viperin catalyzes methionine oxidation to promote protein expression and function of helicases

2019 ◽  
Vol 5 (8) ◽  
pp. eaax1031 ◽  
Author(s):  
Lei Bai ◽  
Jiazhen Dong ◽  
Zhenqiu Liu ◽  
Youliang Rao ◽  
Pinghui Feng ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Methionine Oxidation ◽  
Biological Processes ◽  
Loss Of Function ◽  
Rna Helicases ◽  
Dna And Rna ◽  
Biochemical Studies ◽  
The Stability ◽  
And Function

Helicases play pivotal roles in fundamental biological processes, and posttranslational modifications regulate the localization, function, and stability of helicases. Here, we report that methionine oxidation of representative helicases, including DNA and RNA helicases of viral (ORF44 of KSHV) and cellular (MCM7 and RIG-I) origin, promotes their expression and functions. Cellular viperin, a major antiviral interferon-stimulated gene whose functions beyond host defense remain largely unknown, catalyzes the methionine oxidation of these helicases. Moreover, biochemical studies entailing loss-of-function mutations of helicases and a pharmacological inhibitor interfering with lipid metabolism and, hence, decreasing viperin activity indicate that methionine oxidation potently increases the stability and enzyme activity of these helicases that are critical for DNA replication and immune activation. Our work uncovers a pivotal role of viperin in catalyzing the methionine oxidation of helicases that are implicated in diverse fundamental biological processes.

scholarly journals Synaptic integration of transplanted interneuron progenitor cells into native cortical networks

2016 ◽  
Vol 116 (2) ◽  
pp. 472-478 ◽  
Author(s):  
MacKenzie A. Howard ◽  
Scott C. Baraban
Keyword(s):  
Progenitor Cells ◽  
Neurological Disorders ◽  
Brain Slices ◽  
Pyramidal Cells ◽  
Synaptic Inputs ◽  
Network Function ◽  
Specific Manner ◽  
Therapeutic Mechanisms ◽  
Firing Properties

Interneuron-based cell transplantation is a powerful method to modify network function in a variety of neurological disorders, including epilepsy. Whether new interneurons integrate into native neural networks in a subtype-specific manner is not well understood, and the therapeutic mechanisms underlying interneuron-based cell therapy, including the role of synaptic inhibition, are debated. In this study, we tested subtype-specific integration of transplanted interneurons using acute cortical brain slices and visualized patch-clamp recordings to measure excitatory synaptic inputs, intrinsic properties, and inhibitory synaptic outputs. Fluorescently labeled progenitor cells from the embryonic medial ganglionic eminence (MGE) were used for transplantation. At 5 wk after transplantation, MGE-derived parvalbumin-positive (PV+) interneurons received excitatory synaptic inputs, exhibited mature interneuron firing properties, and made functional synaptic inhibitory connections to native pyramidal cells that were comparable to those of native PV+ interneurons. These findings demonstrate that MGE-derived PV+ interneurons functionally integrate into subtype-appropriate physiological niches within host networks following transplantation.

scholarly journals Set2-Dependent K36 Methylation Is Regulated by Novel Intratail Interactions within H3

2009 ◽  
Vol 29 (24) ◽  
pp. 6413-6426 ◽  
Author(s):  
James N. Psathas ◽  
Suting Zheng ◽  
Song Tan ◽  
Joseph C. Reese
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Transcription Initiation ◽  
Gene Activation ◽  
N Terminus

ABSTRACT Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

Photochemical Degradation of Azo Dyes in the Presence of Hydrogen Peroxide and Hematite under Visible Light Irradiation: Surface Complex Forming and Reaction Mechanism

2011 ◽  
Vol 287-290 ◽  
pp. 1612-1619 ◽  
Author(s):  
Yu Chao Tang ◽  
Xian Huai Huang ◽  
Han Qing Yu ◽  
Wei Hua Li ◽  
Chang Nian Wu
Keyword(s):  
Azo Dyes ◽  
Active Site ◽  
Azo Dye ◽  
Critical Factor ◽  
Chemical Adsorption ◽  
Photochemical Degradation ◽  
Surface Complex ◽  
Degradation Mechanisms ◽  
Decolorization Efficiency ◽  
Visible Irradiation

The photochemical degradation mechanisms of an azo dye Direct Red 4BS and Methyl Orange on hematite in the presence of H2O2 were investigated. The decolorization of azo dyes was attributed to the forming surface complex between specific bond of the dyes and hematite, which facilitate the electron transfer from hematite to azo bond. No mineralization of azo dyes occurred in the presence of visible irradiation, only chromogenic group destroyed in the photo-chemical reaction process. Surface complex between azo dyes and hematite will be destroyed under alkaline solution which suggested the active site or the formed surface complex had been destroyed by OH–. Chemical adsorption of the azo dyes on hematite was critical factor which affect the decolorization efficiency of the photoreaction.

scholarly journals Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

2022 ◽  
Author(s):  
Andrea Piserchio ◽  
Eta A Isiroho ◽  
Kimberly Long ◽  
Amanda L Bohanon ◽  
Eric A Kumar ◽  
...  
Keyword(s):  
Active Site ◽  
Protein Quality ◽  
Regulatory Element ◽  
Structural Basis ◽  
Calmodulin Binding ◽  
Intimate Association ◽  
Biochemical Studies ◽  
Functional Consequences ◽  
Heterodimeric Complex ◽  
Elongation Phase

Translation is a highly energy consumptive process tightly regulated for optimal protein quality and adaptation to energy and nutrient availability. A key facilitator of this process is the α-kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process mechanistically distinct from other calmodulin-dependent kinases. Here we resolve the atomic details of this mechanism through a 2.3 Å crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a spine that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

Engineered Ribosomes for Basic Science and Synthetic Biology

2018 ◽  
Vol 9 (1) ◽  
pp. 311-340 ◽  
Author(s):  
Anne E. d'Aquino ◽  
Do Soon Kim ◽  
Michael C. Jewett
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Active Site ◽  
The Past ◽  
Translation Apparatus ◽  
Biochemical Studies ◽  
Future Challenges ◽  
Fundamental Biology ◽  
Catalytic Capacity ◽  
Bacterial Translation

The ribosome is the cell's factory for protein synthesis. With protein synthesis rates of up to 20 amino acids per second and at an accuracy of 99.99%, the extraordinary catalytic capacity of the bacterial translation machinery has attracted extensive efforts to engineer, reconstruct, and repurpose it for biochemical studies and novel functions. Despite these efforts, the potential for harnessing the translation apparatus to manufacture bio-based products beyond natural limits remains underexploited, and fundamental constraints on the chemistry that the ribosome's RNA-based active site can carry out are unknown. This review aims to cover the past and present advances in ribosome design and engineering to understand the fundamental biology of the ribosome to facilitate the construction of synthetic manufacturing machines. The prospects for the development of engineered, or designer, ribosomes for novel polymer synthesis are reviewed, future challenges are considered, and promising advances in a variety of applications are discussed.

scholarly journals Enzymatic reconstitution of ribosomal peptide backbone thioamidation

2018 ◽  
Vol 115 (12) ◽  
pp. 3030-3035 ◽  
Author(s):  
Nilkamal Mahanta ◽  
Andi Liu ◽  
Shihui Dong ◽  
Satish K. Nair ◽  
Douglas A. Mitchell
Keyword(s):  
Posttranslational Modifications ◽  
Active Site ◽  
Methanogenic Archaea ◽  
Methanosarcina Acetivorans ◽  
Binding Studies ◽  
Peptide Backbone ◽  
Α Subunit ◽  
Essential Enzyme ◽  
Methanopyrus Kandleri

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri. Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

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