Assembly Of Enzyme Research Articles

Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation?

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function.

Hydrodynamic Layer-by-Layer Assembly of Transferable Enzymatic Conductive Nanonetworks for Enzyme-Sticker-Based Contact Printing of Electrochemical Biosensors.

Realizing high-performance electrochemical biosensors in a simple contact-printing-based approach significantly increases the applicability of integrated flexible biosensors. Herein, an enzyme-sticker-based approach that enables flexible and multielectrochemical sensors via simple contact-transfer printing is reported. The enzyme sticker consists of an enzymatic conductive network film and a polymeric support. The enzyme-incorporated nanostructured conductive network showing an efficient electrical coupling was assembled via the hydrodynamic layer-by-layer assembly of redox enzymes, polyelectrolytes, single-walled carbon nanotubes, and a biological glue material, M13 phage. The enzymatic conductive network on a polymeric membrane support was facilely wet contact-transfer printed onto integrated electrode systems by exploiting varying degrees of hydrophilicity displayed by the enzymatic electronic film, polymeric support, and receiving electrodes of the sensor system. The glucose sensors fabricated using the enzyme sticker detected glucose at a concentration of as low as 35 μM and showed high selectivity and stability. Furthermore, a flexible dual-sensor array capable of detecting both glucose and lactate was demonstrated using the versatile enzyme sticker concept. This work presents a new route toward assembling and integrating hybrid nanomaterials with efficient electrochemical coupling for high-performance biosensors and health-monitoring devices as well as for emerging bioelectronics and electrochemical devices.

Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

This study constructed cell‐free glycolytic enzyme systems and compared them to their in vivo functions in Escherichia coli. Under in vitro conditions, flux regulation followed enzyme concentrations and kinetics. In E. coli, only one of the isozymes of phosphofructokinase (PfkA) and fructose‐bisphosphate aldolase (FbaA) facilitate Embden‐Meyerhof‐Parnas (EMP) flux, but under in vitro assays, these isozymes were interchangeable. Additionally, in vitro introduction of the Entner–Doudoroff (ED) pathway improved glycolysis rates, while in vivo overexpression of the ED pathway could not capture significant flux unless its phosphotransferase system (PTS) was knocked out. Lastly, in vivo dynamic 13 C‐experiments revealed that the labeling order of EMP pathway intermediates was not strictly cascade, indicating intracellular metabolites were not well mixed. These enigmatic observations cannot be fully explained by thermodynamics or substrate level regulations. This article supports the long‐time conjecture that EMP enzymes are channeled, and the PTS may be an anchor point to initiate enzyme assemblies. © 2018 American Institute of Chemical Engineers AIChE J, 65: 483–490, 2019

Asymmetric assembly of high-value \u03b1-functionalized organic acids using a biocatalytic chiral-group-resetting process

The preparation of α-functionalized organic acids can be greatly simplified by adopting a protocol involving the catalytic assembly of achiral building blocks. However, the enzymatic assembly of small amino acids and aldehydes to form numerous α-functionalized organic acids is highly desired and remains a significant challenge. Herein, we report an artificially designed chiral-group-resetting biocatalytic process, which uses simple achiral glycine and aldehydes to synthesize stereodefined α-functionalized organic acids. This cascade biocatalysis comprises a basic module and three different extender modules and operates in a modular assembly manner. The engineered Escherichia coli catalysts, which contained different module(s), provide access to α-keto acids, α-hydroxy acids, and α-amino acids with excellent conversion and enantioselectivities. Therefore, this biocatalytic process provides an attractive strategy for the conversion of low-cost achiral starting materials to high-value α-functionalized organic acids.

On the role of the tricarboxylic acid cycle in plant productivity.

The tricarboxylic acid (TCA) cycle is one of the canonical energy pathways of living systems, as well as being an example of a pathway in which dynamic enzyme assemblies, or metabolons, are well characterized. The role of the enzymes have been the subject of saturated transgenesis approaches, whereby the expression of the constituent enzymes were reduced or knocked out in order to ascertain their in vivo function. Some of the resultant plants exhibited improved photosynthesis and plant growth, under controlled greenhouse conditions. In addition, overexpression of the endogenous genes, or heterologous forms of a number of the enzymes, has been carried out in tomato fruit and the roots of a range of species, and in some instances improvement in fruit yield and postharvest properties and plant performance, under nutrient limitation, have been reported, respectively. Given a number of variants, in nature, we discuss possible synthetic approaches involving introducing these variants, or at least a subset of them, into plants. We additionally discuss the likely consequences of introducing synthetic metabolons, wherein certain pairs of reactions are artificially permanently assembled into plants, and speculate as to future strategies to further improve plant productivity by manipulation of the core metabolic pathway.

MITOCHONDRIAL DISEASES (Posters): P.187Mitochondrial disorders with polymerase gamma 1 (POLG1) mutations: a study from tertiary referral centre

Mitochondrial disorders are a heterogeneous and a complex group of neuromuscular disorders resulting from dysfunction of oxidative phosphorylation (OXPHOS). Defects in enzymes encoded by nuclear genes (nDNA) or mitochondrial genome (mtDNA) cause myopathy. The nuclear genome encodes most of the subunits of enzyme complexes, assembly proteins and mtDNA replication, transcription and translation, while, the mitochondrial genome encodes 13subunits of the OXPHOS system, rRNA and tRNA. The cross talk between nDNA and mtDNA is crucial for the cellular regulation of mtDNA integrity, copy number and mitochondrial protein production. POLG1, a nuclear gene essential for mtDNA replication encodes for the catalytic subunit of mitochondrial polymerase gamma. Mutations in POLG1 results in a spectrum of clinical phenotypes. In this study, 910 patients seen between 2002-2017, clinically fulfilling the modified Walkers criteria who underwent skeletal muscle biopsy were analyzed. Long range PCR done on 630/910 cases, revealed multiple deletions in 71. We have sequenced intron-exon boundaries of POLG1 in 351 cases (71 cases with multiple deletions and 280 cases where deletions could not done). Mutations were identified in 18/351(5%) patients. The most common clinical phenotype noted in these 18 cases was CPEO. There were 9 males and 9 females with age at onset ranging from 2 -20 years. Morphologically, muscle biopsy revealed characteristic ragged red fibres with ultrastructural evidence of abnormal mitochondria in 8 cases. Respiratory chain analysis revealed a multiple complex deficiency in 8/18. The reported mutations identified include p.L304R (n=10), p.A682T(n=1), p.R1097G (n=1), p.E1143G (n=1), p.H110Y & p.E1143G (n=1), p.R1187W (n=1), p.R627Q (n=1). In addition, two novel mutations p.Q49H and p.V1106A were noted. In our cohort, a p.L304R mutation was more prevalent and seen in younger age group. Detailed clinical, biochemical, pathological and genetics findings will be presented.

Redox-switchable siderophore anchor enables reversible artificial metalloenzyme assembly

Artificial metalloenzymes that contain protein-anchored synthetic catalysts are attracting increasing interest. An exciting, but still unrealized advantage of non-covalent anchoring is its potential for reversibility and thus component recycling. Here we present a siderophore–protein combination that enables strong but redox-reversible catalyst anchoring, as exemplified by an artificial transfer hydrogenase (ATHase). By linking the iron(iii)-binding siderophore azotochelin to an iridium-containing imine-reduction catalyst that produces racemic product in the absence of the protein CeuE, but a reproducible enantiomeric excess if protein bound, the assembly and reductively triggered disassembly of the ATHase was achieved. The crystal structure of the ATHase identified the residues involved in high-affinity binding and enantioselectivity. While in the presence of iron(iii), the azotochelin-based anchor binds CeuE with high affinity, and the reduction of the coordinated iron(iii) to iron(ii) triggers its dissociation from the protein. Thus, the assembly of the artificial enzyme can be controlled via the iron oxidation state.

Modern approaches to artificial gene synthesis: aspects of oligonucleotide synthesis, enzymatic assembly, sequence verification and error correction

Synthetic biology is a rapidly developing field aimed at engineering of biological systems with predictable properties. Synthetic biology accumulates the achievements of modern biological sciences, programming and computational model­ing as well as engineering technologies for creation of biologi­cal objects with user-defined properties. Evolution of synthetic biology has been marked by a number of technological developments in each of the mentioned fields. Thus, significant reduction in cost of DNA sequencing has provided an easy access to large amounts of data on the genetic sequences of various organisms, and decreased the price of the DNA sequence synthesis, which, analogous to Moore’s law, resulted in an opportunity to create a lot of potential genes without the time – consuming and labor – intensive traditional methods of molecular biology. Development of system biology has allowed forming a deeper understanding of the functions and relationship of natural biological models, as well as of the computational models describing processes at the cell and system levels. Combination of these factors has created an op­portunity for conscious changes of natural biological systems. In this review the modern approaches to oligonucleotide gene assembly synthesis are discussed, including such aspects as protocols for gene assembly, sequence verification, error cor­rection and further applications of synthesized genes.

The small molecule drug diminazene aceturate inhibits liver injury and biliary fibrosis in mice

There is no established medical therapy to treat biliary fibrosis resulting from chronic inflammation in the biliary tree. We have recently shown that liver-specific over-expression of angiotensin converting enzyme 2 (ACE2) of the renin angiotensin system (RAS) ameliorated liver fibrosis in mice. Diminazene aceturate (DIZE), a small molecule drug approved by the US Food and Drug Administration, which is used to treat human trypanosomiasis, has been shown to have antifibrotic properties by enhancing ACE2 activity. In this study we sought to determine the therapeutic potential of DIZE in biliary fibrosis using bile duct ligated and multiple drug resistant gene-2 knockout mice. Additionally, human hepatic stellate (LX-2) and mouse Kupffer (KUP5) cell lines were used to delineate intracellular pathways. DIZE treatment, both in vivo and in vitro, markedly inhibited the activation of fibroblastic stellate cells which was associated with a reduced activation of Kupffer cells. Moreover, DIZE-inhibited NOX enzyme assembly and ROS generation, activation of profibrotic transcription factors including p38, Erk1/2 and Smad2/3 proteins and proinflammatory and profibrotic cytokine release. These changes led to a major reduction in biliary fibrosis in both models without affecting liver ACE2 activity. We conclude that DIZE has a potential to treat biliary fibrosis.

An Activity Switch in Human Telomerase Based on RNA Conformation and Shaped by TCAB1

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerasereverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.

Self-Assembled Multimeric-Enzyme Nanoreactor for Robust and Efficient Biocatalysis.

The construction of artificial multienzyme nanodevices with desired spatial arrangements have shown great promise for improving the overall performance of targeted enzyme cascades. However, it is a challenge to rationally design and construct multiple oligomeric enzyme assemblies that can be used as stable and reusable catalysts. Herein, we report a novel approach to rapidly achieve ultrastable multimeric enzyme nanoclusters (MENCs) based on enzymes property of oligomerization and the SpyTag/SpyCatcher system of orthogonally reactive split peptides. The SpyCatcher peptide and its binding partner SpyTag were fused to a dimeric cytochrome P450 monooxygenase mutant (P450BM3m) and a tetrameric glucose dehydrogenase (GDH), respectively. The fusion proteins self-assembled into the MENCs, forming a covalently coupled supramolecular multienzyme nanodevices that facilitated NADPH regeneration and converted indole into a pigment indigo. We investigated the morphology of the MENCs and found these multimeric enzymes assembled into two-dimensional layerlike nanoscale architecture, ranging from a few to several hundred square microns in size. Importantly, enzymatic analysis revealed that the MENCs not only increased the initial rate by more than three times for the indigo synthesis, but also achieved significant improvements on stability and reusability compared to unassembled enzyme mixtures. This work demonstrates a versatile and efficient strategy to construct stable and multifunctional biocatalysts with potential applications in metabolic engineering and synthetic biology.

Biofabricating Functional Soft Matter Using Protein Engineering to Enable Enzymatic Assembly.

Biology often provides the inspiration for functional soft matter, but biology can do more: it can provide the raw materials and mechanisms for hierarchical assembly. Biology uses polymers to perform various functions, and biologically derived polymers can serve as sustainable, self-assembling, and high-performance materials platforms for life-science applications. Biology employs enzymes for site-specific reactions that are used to both disassemble and assemble biopolymers both to and from component parts. By exploiting protein engineering methodologies, proteins can be modified to make them more susceptible to biology's native enzymatic activities. They can be engineered with fusion tags that provide (short sequences of amino acids at the C- and/or N- termini) that provide the accessible residues for the assembling enzymes to recognize and react with. This "biobased" fabrication not only allows biology's nanoscale components (i.e., proteins) to be engineered, but also provides the means to organize these components into the hierarchical structures that are prevalent in life.

Extramitochondrial Assembly of Mitochondrial Targeting Signal Disrupted Mitochondrial Enzyme Aldehyde Dehydrogenase

Supramolecular assembly of metabolic enzymes has been studied both in vivo and in vitro for nearly a decade. Experimental evidence has suggested a close relationship between enzymatic activity and enzyme assembly/disassembly. However, most cases were studied with the cytosolic enzymes. Here, I report the evidence for a mitochondrial enzyme with its ability in forming visible intracellular structures. By removing the mitochondrial targeting sequence, yeast mitochondrial enzyme aldehyde dehydrogenase (Ald4p) exhibits reversible supramolecular assembly in the cytoplasm, thus creating a useful system for further characterization of the regulatory factors that modulate the assembly/disassembly of this mitochondrial enzyme.

Studying protein‐protein interactions in fatty acid and polyketide biosynthetic pathways via site‐specific vibrational spectroscopy

By virtue of their structural diversity and striking potency, natural products have long been used as therapeutic agents. Natural products are biosynthesized by assemblies of enzymes encoded in gene clusters. Repurposing the natural product biosynthetic repertoire to assemble “unnatural” natural products would have profound implications for human health. Our current lack of understanding of how these biosynthetic enzymes operate at a molecular level, which is partially attributed to the lack of tools that can capture the weak and transient interactions of the enzyme assemblies, stymies such engineering endeavors. Our lab previously showed that labeling acyl carrier proteins, which are central hubs of natural product assemblies, with a thiocyanate (SCN) vibrational probe provides an innovative method to study the proteins' conformational dynamics and interactions with ketosynthases. In this work, we evaluate the general utility of the probe by assessing ACP‐SCN interactions with other partner enzymes. The SCN probe was loaded onto the 4′‐phosphopantetheine (Ppant) arm of ACPs involved in fatty acid and actinorhodin type II polyketide biosynthetic pathways, AcpP and actACP, respectively. Changes in the CN stretching frequency were monitored as the ACPs were titrated with partner enzymes, 3‐hydroxyacyl‐ACP dehydrase (FabA) and actinorhodin polyketide ketoreductase (actKR). Upon a productive interaction of the ACP with its respective partner enzyme, the CN absorption band shifts to lower wavenumbers, which could correlate to the probe accessing the partner enzyme's hydrophobic catalytic active site. In light of these findings, the SCN probe appears to report on a wide range of ACP‐partner protein interactions. As a result, the probe could be a useful tool for future engineering studies.Support or Funding InformationWe would like to acknowledge an NIH Award #CHE‐1652424 to Louise K. Charkoudian as a source of funding for this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Consequences of Cytochrome c Oxidase Assembly Defects for the Yeast Stationary Phase

Functional assembly of cytochrome c oxidase (COX) is essential for an intact mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase remain largely unexplored. Stationary phase cultures are a good model for terminally differentiated cells in humans, such as neurons. Our goal is to define the physiological changes in mitochondrial respiration and oxidative stress between exponential and stationary phases in a respiratory competent strain and a set of COX assembly mutants with defects in a variety of aspects related to enzyme assembly. These results further our understanding of mitochondrial changes in terminally differentiated cells and aging cells. In this study, we show that a wild‐type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX, which is supported by a decrease in protein levels of several COX assembly factors. Wild‐type cells also have a disrupted mitochondrial reticulum during stationary phase. Loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which we show is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Specifically, differences in distribution of Sod1 between exponential and stationary phases give insight on location of reactive oxygen species (ROS) in the cell. Our results suggest that mitochondria may not be a major source of ROS at stationary phase in cells lacking an intact respiratory chain. Overall, we have identified fundamental differences between exponential and stationary phase mitochondria in respiratory competent yeast and have defined two distinct phenotypes, decreased levels of mitochondrial proteins and less mitochondrial oxidative damage, in cells with a defective respiratory chain. These findings suggest that COX deficiencies are associated with broader cellular phenotypes in terminally differentiated cells in humans.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NADPH oxidase 2 knockout prevents chronic intermittent hypoxia induced sternohyoid muscle weakness in adult male mice

Obstructive sleep apnoea syndrome (OSAS) is the most prevalent form of sleep disordered breathing (SDB) which affects 1 in 5 people worldwide. Chronic intermittent hypoxia (CIH) is a hallmark feature of SDB as a consequence of repetitive upper airway occlusions in patients during sleep. These recurring hypoxia‐reoxygenation cycles result in the overproduction of reactive oxygen species (ROS), ultimately yielding a state of oxidative stress. Excessive levels of ROS have been associated with aberrant plasticity at multiple levels of the respiratory system including impaired upper airway muscle function. However, there is a paucity of information regarding the molecular mechanisms underlying these effects. The NADPH oxidase (NOX) family of enzymes have been linked to a multitude of CIH induced morbidities in a range of organs and systems with NOX subunits also having been identified in skeletal muscle.A mouse model of CIH was generated by the cycling of gas from normoxia (21% O2) for 210 seconds to hypoxia (5% O2 at the nadir) for 90 seconds 8 hr/day for 2 weeks during light hours. 10–11 week old male mice (C57BL/6J) were randomly assigned to one of 3 groups: Normoxic controls (sham; n=24), CIH exposed (n=24), and CIH + Apocynin (2 mM; n=24) treated throughout the gas exposure. 10–11 week old NOX2 null (B6.129S‐Cybbtm1Din/J) male mice were also assigned to a sham (n=12) or CIH (n=12) group. Following this, sternohyoid muscle contractile function was examined ex vivo. Gene expression of the entire family of NOX enzymes was examined by RT‐PCR. Western blot was used to quantify NOX2 and NOX4 protein expression. NOX enzyme activity was determined using a NOX activity assay. Data were expressed as mean±SD and were statistically compared by unpaired Student t‐test or two‐way ANOVA with Bonferroni post‐hoc test.2 weeks of exposure to CIH resulted in a significant decrease of ~45% in the peak specific force (Fmax) of the sternohyoid muscle when compared to normoxic controls (sham). CIH resulted in no significant alteration to the gene expression of any members of the NOX family of enzymes when compared to sham controls. There was no significant difference in NOX2 or NOX4 protein expression between CIH and sham groups. However, CIH exposure resulted in a significant increase in sternohyoid NOX enzyme activity compared to shams. Treatment with Apocynin (2 mM) throughout the 2 week CIH exposure prevented sternohyoid muscle weakness, restoring force to levels equivalent to controls. Furthermore, NOX2 knockout prevented CIH induced sternohyoid muscle weakness.Adult male C57BL/6J mice show signs of profound sternohyoid muscle dysfunction following exposure to 2 weeks of CIH when compared with sham animals. This occurs without alteration to the gene or protein expression of various NOX subunits. Increased NOX activity suggests a CIH induced increase in NOX enzyme assembly rather than abundance. Administration of the putative NOX inhibitor, Apocynin, prevents sternohyoid muscle weakness. NOX2 knockout also entirely prevents sternohyoid muscle weakness confirming its specific role in CIH induced muscle dysfunction. The striking muscle phenotype in the C57 mouse and its prevention in the absence of NOX2 provides a novel and robust platform for the study of mechanisms of hypoxia‐related muscle dysfunction, which may have relevance to respiratory conditions characterised by CIH, such as sleep apnoea.Support or Funding InformationDepartment of Physiology, University College Cork, Cork, IrelandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vivo Quantitative Monitoring of Subunit Stoichiometry for Metabolic Complexes.

Metabolic pathways often employ assemblies of individual enzymes to facilitate substrate channeling to improve thermodynamic efficiency and confer pathway directionality. It is often assumed that subunits to multienzyme complexes are coregulated and accumulate at fixed levels in vivo, reflecting complex stoichiometry. Such assumptions can be experimentally tested using modern tandem mass spectrometry, and herein we describe such an approach applied toward an important metabolic complex. The committed step of de novo fatty acid synthesis in the plastids of most plants is catalyzed by the multienzyme, heteromeric acetyl-CoA carboxylase (hetACCase). This complex is composed of four catalytic subunits and a recently discovered regulatory subunit resembling the biotin carboxyl carrier protein but lacking the biotinylation motif necessary for activity. To better understand this novel form of regulation, a targeted tandem mass-spectrometry-based assay was developed to absolutely quantify all subunits to the Arabidopsis thaliana hetACCase. After validation against pure, recombinant protein, this multiplexed assay was used to quantify hetACCase subunits in siliques in various stages of development. Quantitation provided a developmental profile of hetACCase and BADC protein expression that supports a recently proposed regulatory mechanism for hetACCase and demonstrates a promising application of targeted mass spectrometry for in vivo analysis of protein complexes.

Controllable Assembly of Enzymes for Multiplexed Lab‐on‐a‐Chip Bioassays with a Tunable Detection Range

AbstractMultiplexed analysis of molecules with different concentrations requires assays with a tunable detection range. A strategy is outlined that uses click chemistry to assemble horseradish peroxidase in a controlled fashion to generate enzyme assemblies as probes for multiplexed bioassays. This controllable assembly of enzymes on detection antibodies allows for lab‐on‐a‐chip immunoassays with a tunable detection range from pg mL−1 to μg mL−1. Simultaneous, multiplexed bioassays of clinically relevant inflammatory biomarkers in serum are demonstrated in one lab‐on‐a‐chip format, with a limit of detection of 0.47 pg mL−1 for interleukin‐6, 2.6 pg mL−1 for procalcitonin, and 40 ng mL−1 for C‐reactive protein. This controlled assembly technique provides a multiplexed platform for simultaneous and quantitative analyses of both low‐abundance and high‐abundance biomarkers with a broad detection range, which holds great promise as a point‐of‐care platform for biomedical diagnostics.

Controllable Assembly of Enzymes for Multiplexed Lab‐on‐a‐Chip Bioassays with a Tunable Detection Range

Multiplexed analysis of molecules with different concentrations requires assays with a tunable detection range. A strategy is outlined that uses click chemistry to assemble horseradish peroxidase in a controlled fashion to generate enzyme assemblies as probes for multiplexed bioassays. This controllable assembly of enzymes on detection antibodies allows for lab-on-a-chip immunoassays with a tunable detection range from pg mL-1 to μg mL-1 . Simultaneous, multiplexed bioassays of clinically relevant inflammatory biomarkers in serum are demonstrated in one lab-on-a-chip format, with a limit of detection of 0.47 pg mL-1 for interleukin-6, 2.6 pg mL-1 for procalcitonin, and 40 ng mL-1 for C-reactive protein. This controlled assembly technique provides a multiplexed platform for simultaneous and quantitative analyses of both low-abundance and high-abundance biomarkers with a broad detection range, which holds great promise as a point-of-care platform for biomedical diagnostics.

Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150–400 kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.