scholarly journals The AAA+ ATPase p97, a cellular multitool

2017 ◽  
Vol 474 (17) ◽  
pp. 2953-2976 ◽  
Author(s):  
Lasse Stach ◽  
Paul S. Freemont
Keyword(s):  
Atp Hydrolysis ◽  
Current Status ◽  
Cellular Functions ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Proteotoxic Stress ◽  
Binding Domains ◽  
Wide Range ◽  
Aaa Atpases ◽  
Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

scholarly journals Phosphatidylinositol-3,4,5-Triphosphate and Cellular Signaling: Implications for Obesity and Diabetes

2015 ◽  
Vol 35 (4) ◽  
pp. 1253-1275 ◽  
Author(s):  
Prasenjit Manna ◽  
Sushil K. Jain
Keyword(s):  
Metabolic Diseases ◽  
Specific Binding ◽  
Cell Metabolism ◽  
Effector Proteins ◽  
Current Status ◽  
Cellular Functions ◽  
Binding Domains ◽  
Obesity And Diabetes ◽  
Impaired Metabolism ◽  
Wide Range

Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is one of the most important phosphoinositides and is capable of activating a wide range of proteins through its interaction with their specific binding domains. Localization and activation of these effector proteins regulate a number of cellular functions, including cell survival, proliferation, cytoskeletal rearrangement, intracellular vesicle trafficking, and cell metabolism. Phosphoinositides have been investigated as an important agonist-dependent second messenger in the regulation of diverse physiological events depending upon the phosphorylation status of their inositol group. Dysregulation in formation as well as metabolism of phosphoinositides is associated with various pathophysiological disorders such as inflammation, allergy, cardiovascular diseases, cancer, and metabolic diseases. Recent studies have demonstrated that the impaired metabolism of PtdIns(3,4,5)P3 is a prime mediator of insulin resistance associated with various metabolic diseases including obesity and diabetes. This review examines the current status of the role of PtdIns(3,4,5)P3 signaling in the regulation of various cellular functions and the implications of dysregulated PtdIns(3,4,5)P3 signaling in obesity, diabetes, and their associated complications.

scholarly journals Cooperative subunit dynamics modulate p97 function

2018 ◽  
Vol 116 (1) ◽  
pp. 158-167 ◽  
Author(s):  
Rui Huang ◽  
Zev A. Ripstein ◽  
John L. Rubinstein ◽  
Lewis E. Kay
Keyword(s):  
Bound State ◽  
Nmr Studies ◽  
Biologically Relevant ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Allosteric Communication ◽  
Functional Dynamics ◽  
Nmr Study ◽  
Wide Range

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

scholarly journals Distinct segregation patterns of yeast cell-peripheral proteins uncovered by a method for protein segregatome analysis

2019 ◽  
Vol 116 (18) ◽  
pp. 8909-8918 ◽  
Author(s):  
Shinju Sugiyama ◽  
Motomasa Tanaka
Keyword(s):  
Wall Stress ◽  
Cell Periphery ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Binding Domains ◽  
Peripheral Proteins ◽  
Flow Through ◽  
Membrane Spanning ◽  
Mother Cells ◽  
Er Membrane

Protein segregation contributes to various cellular processes such as polarization, differentiation, and aging. However, the difficulty in global determination of protein segregation hampers our understanding of its mechanisms and physiological roles. Here, by developing a quantitative proteomics technique, we globally monitored segregation of preexisting and newly synthesized proteins during cell division of budding yeast, and identified crucial domains that determine the segregation of cell-peripheral proteins. Remarkably, the proteomic and subsequent microscopic analyses demonstrated that the flow through the bud neck of the proteins that harbor both endoplasmic reticulum (ER) membrane-spanning and plasma membrane (PM)-binding domains is not restricted by the previously suggested ER membrane or PM diffusion barriers but by septin-mediated partitioning of the PM-associated ER (pmaER). Furthermore, the proteomic analysis revealed that although the PM-spanning t-SNARE Sso2 was retained in mother cells, its paralog Sso1 unexpectedly showed symmetric localization. We found that the transport of Sso1 to buds was required for enhancement of polarized cell growth and resistance to cell-wall stress. Taken together, these data resolve long-standing questions about septin-mediated compartmentalization of the cell periphery, and provide new mechanistic insights into the segregation of cell-periphery proteins and their cellular functions.

scholarly journals Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1403
Author(s):  
Maryia Karpiyevich ◽  
Katerina Artavanis-Tsakonas
Keyword(s):  
Mammalian Cells ◽  
Dynamic Balance ◽  
Fine Tuning ◽  
Protozoan Parasites ◽  
Cellular Functions ◽  
Parasitic Protozoa ◽  
Distinctive Features ◽  
Cellular Processes ◽  
Wide Range ◽  
Enzymatic Machinery

Post-translational protein regulation allows for fine-tuning of cellular functions and involves a wide range of modifications, including ubiquitin and ubiquitin-like modifiers (Ubls). The dynamic balance of Ubl conjugation and removal shapes the fates of target substrates, in turn modulating various cellular processes. The mechanistic aspects of Ubl pathways and their biological roles have been largely established in yeast, plants, and mammalian cells. However, these modifiers may be utilised differently in highly specialised and divergent organisms, such as parasitic protozoa. In this review, we explore how these parasites employ Ubls, in particular SUMO, NEDD8, ATG8, ATG12, URM1, and UFM1, to regulate their unconventional cellular physiology. We discuss emerging data that provide evidence of Ubl-mediated regulation of unique parasite-specific processes, as well as the distinctive features of Ubl pathways in parasitic protozoa. We also highlight the potential to leverage these essential regulators and their cognate enzymatic machinery for development of therapeutics to protect against the diseases caused by protozoan parasites.

scholarly journals Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry

2020 ◽  
Vol 19 (12) ◽  
pp. 1997-2014
Author(s):  
Yadong Yu ◽  
Haichuan Liu ◽  
Zanlin Yu ◽  
H. Ewa Witkowska ◽  
Yifan Cheng
Keyword(s):  
Protein Unfolding ◽  
Atp Hydrolysis ◽  
Mass Measurement ◽  
Site Occupancy ◽  
Internal Standard ◽  
Large Family ◽  
Nucleotide Binding ◽  
Native Mass Spectrometry ◽  
Aaa Atpase ◽  
Aaa Atpases

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

scholarly journals Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast

2020 ◽  
Author(s):  
Nathaniel L. Hepowit ◽  
Kevin N. Pereira ◽  
Jessica M. Tumolo ◽  
Walter J. Chazin ◽  
Jason A. MacGurn
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Membrane Trafficking ◽  
Stress Responses ◽  
Protein Quality ◽  
Oxidative Stress Response ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Proteotoxic Stress ◽  
Substrate Proteins

ABSTRACTUbiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in Saccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.

scholarly journals Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

2015 ◽  
Vol 112 (20) ◽  
pp. 6371-6376 ◽  
Author(s):  
Matthew P. Nicholas ◽  
Florian Berger ◽  
Lu Rao ◽  
Sibylle Brenner ◽  
Carol Cho ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Atp Hydrolysis ◽  
Cytoplasmic Dynein ◽  
Atp Binding ◽  
Mechanical Tension ◽  
Main Site ◽  
Aaa Atpase ◽  
C Terminus ◽  
Directed Motility ◽  
Aaa Atpases

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end–directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1–4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein’s motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate “gating” of AAA1 function by AAA3. When tension is absent or applied via dynein’s C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to “open” the gate. These results elucidate the mechanisms of dynein–MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

scholarly journals Mechanisms, regulation and consequences of protein SUMOylation

2010 ◽  
Vol 428 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Kevin A. Wilkinson ◽  
Jeremy M. Henley
Keyword(s):  
Protein Function ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Wide Range ◽  
Lysine Residues ◽  
Ubiquitination Pathway ◽  
Regulate Protein ◽  
Substrate Proteins ◽  
Protein Sumoylation

The post-translational modification SUMOylation is a major regulator of protein function that plays an important role in a wide range of cellular processes. SUMOylation involves the covalent attachment of a member of the SUMO (small ubiquitin-like modifier) family of proteins to lysine residues in specific target proteins via an enzymatic cascade analogous to, but distinct from, the ubiquitination pathway. There are four SUMO paralogues and an increasing number of proteins are being identified as SUMO substrates. However, in many cases little is known about how SUMOylation of these targets is regulated. Compared with the ubiquitination pathway, relatively few components of the conjugation machinery have been described and the processes that specify individual SUMO paralogue conjugation to defined substrate proteins are an active area of research. In the present review, we briefly describe the SUMOylation pathway and present an overview of the recent findings that are beginning to identify some of the mechanisms that regulate protein SUMOylation.

Cellular consequences of inositol depletion

2009 ◽  
Vol 37 (5) ◽  
pp. 1099-1103 ◽  
Author(s):  
Rania M. Deranieh ◽  
Miriam L. Greenberg
Keyword(s):  
Bipolar Disorder ◽  
Inositol Phosphates ◽  
Signalling Pathway ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Wide Range ◽  
Therapeutic Mechanism

The inositol-depletion hypothesis was suggested to explain the therapeutic mechanism of mood-stabilizing drugs. Focus was previously on the phosphatidylinositol signalling pathway and on the regulatory roles of Ins(3,4,5)P3 and DAG (diacylglycerol). Recent findings indicate that inositol and inositol-containing molecules, including phosphoinositides and inositol phosphates, have signalling and regulatory roles in many cellular processes. This suggests that depleting inositol may lead to perturbation of a wide range of cellular functions, at least some of which may be associated with bipolar disorder.

scholarly journals ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

2008 ◽  
Vol 364 (1514) ◽  
pp. 247-255 ◽  
Author(s):  
Daniella Muallem ◽  
Paola Vergani
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Functional Difference ◽  
Transmembrane Conductance Regulator ◽  
Cellular Functions ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

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