Critical Functional Properties Research Articles

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Enhancing Functional Properties and Protein Structure of Almond Protein Isolate Using High-Power Ultrasound Treatment.

The suitability of a given protein for use in food products depends heavily on characteristics such as foaming capacity, emulsifiability, and solubility, all of which are affected by the protein structure. Notably, protein structure, and thus characteristics related to food applications, can be altered by treatment with high-power ultrasound (HUS). Almonds are a promising source of high-quality vegetable protein for food products, but their physicochemical and functional properties remain largely unexplored, limiting their current applications in foods. Here, we tested the use of HUS on almond protein isolate (API) to determine the effects of this treatment on API functional properties. Aqueous almond protein suspensions were sonicated at varying power levels (200, 400, or 600 W) for two durations (15 or 30 min). The molecular structure, protein microstructure, solubility, and emulsifying and foaming properties of the resulting samples were then measured. The results showed that HUS treatment did not break API covalent bonds, but there were notable changes in the secondary protein structure composition, with the treated proteins showing a decrease in α-helices and β-turns, and an increase in random coil structures as the result of protein unfolding. HUS treatment also increased the number of surface free sulfhydryl groups and decreased the intrinsic fluorescence intensity, indicating that the treatment also led to alterations in the tertiary protein structures. The particle size in aqueous suspensions was decreased in treated samples, indicating that HUS caused the dissociation of API aggregates. Finally, treated samples showed increased water solubility, emulsifying activity, emulsifying stability, foaming capacity, and foaming stability. This study demonstrated that HUS altered key physicochemical characteristics of API, improving critical functional properties including solubility and foaming and emulsifying capacities. This study also validated HUS as a safe and environmentally responsible tool for enhancing desirable functional characteristics of almond proteins, promoting their use in the food industry as a high-quality plant-based protein.

The endothelium in lung fibrosis: a core signaling hub in disease pathogenesis?

Pulmonary fibrosis is a progressive chronic lung disease characterized by excessive deposition of extracellular matrix and structural destruction, associated with a severe five year mortality rate. The onset of the disease is thought to be triggered by chronic damage to the alveolar epithelium. Since the pulmonary endothelium is an important component of the alveolar-capillary niche, it is also affected by the initial injury. In addition to ensuring proper gas exchange, the endothelium has critical functional properties, including regulation of vascular tone, inflammatory responses, coagulation and maintenance of vascular homeostasis and integrity. Recent single-cell analyses have shown that shifts in endothelial cell subtypes occur in pulmonary fibrosis. Furthermore, the increased vascular remodeling associated with pulmonary fibrosis leads to deteriorated outcomes for patients, underscoring the importance of the vascular bed in pulmonary fibrosis. To date, the causes and consequences of endothelial and vascular involvement in pulmonary fibrosis are poorly understood. Therefore, it is of great importance to investigate the involvement of endothelial cells and the vascular system in the pathogenesis of the disease. In this review, we will outline the current knowledge on the role of the pulmonary vasculature in pulmonary fibrosis, in terms of abnormal cellular interactions, hyperinflammation, vascular barrier disorders and an altered basement membrane composition. Finally, we will summarize recent advances in extensive therapeutic research and discuss the significant value of novel therapies targeting the endothelium.

Conditioned medium of mesenchymal stem cells pretreated with H2O2 promotes intestinal mucosal repair in acute experimental colitis

Mesenchymal stem cells (MSCs) are a new therapeutic strategy for inflammatory bowel disease (IBD), and their efficacy has been widely recognized. However, there are still some challenges in cell therapy, including stable cell passage, laboratory conditions for cell culture, high-cost burden, and poor transplantation. The conditioned medium (CM) of MSCs is considered be an excellent alternative to cell transplantation, but the paracrine group in MSC-CM is limited in variety and low in concentration, which cannot meet the therapeutic needs of injured tissues and needs to be optimized. Pretreatment with low concentration of hydrogen peroxide (H2O2) can not only protect cells from oxidative damage, but also play a role similar to growth factors and regulate the physiological function of stem cells, to obtain an improved conditioned medium. To determine the optimal protocol for pretreatment of MSCs with H2O2, and to study the efficacy and potential mechanism of MSC-CM pretreated with H2O2 on Dextran Sulfate Sodium (DSS)-induced acute experimental colitis. MSCs were exposed to different concentrations of H2O2, and the optimal H2O2 pretreatment conditions were determined by evaluating their critical cell functional properties. H2O2-pretreated MSC-CM was transplanted into experimental mouse colitis by enema at 2, 4, and 6 days in modeling, and the changes of colonic tissue structure, the levels of inflammation and oxidative stress, the molecular changes of Nrf2/Keap1/ARE axis, and the related indicators of apoptosis in colonic epithelial cells were observed in each group. In vitro, Pretreated MSCs with 25 μM H2O2 significantly enhanced cell proliferation, migration, and survival, but had no effect on apoptosis. In vivo, MSC-CM treatment decreased apoptosis and extracellular matrix deposition, and maintained the mechanical barrier and permeability of colonic epithelial cells in experimental mouse colitis. Mechanistically, H2O2-pretreated MSC-CM against reactive oxygen species (ROS) production and MDA generation, accompanied by increases in components of the enzymatic antioxidant system includes SOD, CAT, GSH-PX, and T-AOC, which is through the up-regulation of the Nrf2, HO-1, and NQO-1 antioxidant genes. Our data confirmed that 25 μM H2O2 pretreated MSC-CM treatment could effectively improve intestinal mucosal repair in experimental colitis, which may be achieved by activating Nrf2/Keap1/ARE pathway.

Influence of acidification or alkalization followed by neutralization on dissolution and acid gelation ability of MPI

Rehydration of milk protein isolate (MPI) powder in water is poor which requires more than 24 h, however it is greatly influenced by solution pH. This study analysed the rehydration of MPI powder in acidic solution followed by neutralization (CN), and in alkalized solution followed by neutralization (NC), and how these pH adjustments impact the dissolution ability and functionality of the MPI. Besides, C’N’ and N’C’ are prepared by the above neutralization sequence with increasing rehydration time. Acid gelation was chosen as an example of critical functional properties. The results showed that applying alkalization (performed by Na-carbonate) as the first step prior to neutralization was superior to acidification (using citric acid) in terms of both dissolution and gelation. MPI powder rehydrated in distilled water gelled very poorly, and this was due to slow dissolution, while neutralized MPI (CN, C’N’, NC and N’C’) produced much stronger gels, in-part due to rapid dissolution and disrupted casein micelle. NC and N’C’ exhibited better gelation characteristics than CN and C’N’ overall, showing much higher elasticity values and earlier gelation point. These were also compared with the rehydration of sodium caseinate, which displayed more rapid dissolution and consequently faster gelation; however, the gelation characteristics became similar to NC over time. N’C’ with longer rehydration time could display further improved gelation characteristics owning higher elasticity than that of SC and SC’. In addition, the neutralized MPI was able to tolerate over-acidification during the acid gelation process.

Endothelial EphB4 maintains vascular integrity and transport function in adult heart.

The homeostasis of heart and other organs relies on the appropriate provision of nutrients and functional specialization of the local vasculature. Here, we have used mouse genetics, imaging and cell biology approaches to investigate how homeostasis in the adult heart is controlled by endothelial EphB4 and its ligand ephrin-B2, which are known regulators of vascular morphogenesis and arteriovenous differentiation during development. We show that inducible and endothelial cell-specific inactivation of Ephb4 in adult mice is compatible with survival, but leads to rupturing of cardiac capillaries, cardiomyocyte hypertrophy, and pathological cardiac remodeling. In contrast, EphB4 is not required for integrity and homeostasis of capillaries in skeletal muscle. Our analysis of mutant mice and cultured endothelial cells shows that EphB4 controls the function of caveolae, cell-cell adhesion under mechanical stress and lipid transport. We propose that EphB4 maintains critical functional properties of the adult cardiac vasculature and thereby prevents dilated cardiomyopathy-like defects.

A scale-free analysis of the HIV-1 genome demonstrates multiple conserved regions of structural and functional importance.

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 5′ end of env.

Achieving coalesced breathability, mechanical and shape memory properties of collagen fibrous matrix through complexing with chromium (III)

Protein materials evolved in nature for millions of years can display critical structural and functional properties which is difficult for synthetic polymers to surpass. Hence, the combination of multiple components or hierarchies from natural source through a facile method may stand out as a forerunner to materials design and application. Inspired from metalloprotein, this study reported an organic-metal biosystem based on chromium (III) complexed collagen fibrous matrix (Cr-CFM). The obtained Cr-CFM retains inherent conformation of collagen and its fiber morphology in the skin. Compared with pristine CFM, Cr-CFM showed enhanced waterproofness and breathability. Due to coexistence of hydrogen bonds and newly introduced chromium (III) complexing linkages, Cr-CFM exhibited water-adaptive mechanical property. In this organic-metal biosystem, hydrogen bonds show reversible cleavage-reformation under water interference while chromium (III) complexing linkages remains stable. Such combination of “switch” and “netpoint” facilitated water responsive shape memory ability of Cr-CFM with shape fixation and recovery reaching over 80%. The reveal of relationship between structure and coalesced properties of Cr-CFM herein is believed to promote progress of smart protein materials and give inspirations to synthetic polymers.

Critical functional properties of defatted peanut meal produced by aqueous extraction and conventional methods.

The functional properties of the defatted peanut meal produced by aqueous extraction, solvent extraction and cold screw pressing followed by solvent extraction were studied. Good gelling property, color and nitrogen dispersibility as well as the high protein content of the defatted peanut meals produced by these methods were verified to be the critical functional properties for their high value of application to the food industry (e.g. production of ham sausages or hotdog sausages) though they are not suggested to be applied to the foods needing good oil binding capacity, emulsifying activity or stability and foaming capacity or stability. These results should provide valuable reference data for the application of defatted peanut meal to the food industry, the correct selection of processing method of peanut kernels which should be pursued by manufacturers and the determination of research direction that peanut processing technologists should be interested in.

197 - Functional & economic comparability of Xeno-free stem cell bioprocess system

Human Mesenchymal Stem Cells (hMSCs) are key raw material in Regenerative Medicine and are widely used for therapeutics, engineered tissues, and medical devices. Major challenges for manufacturing this key raw material lies in the culture time, cost and media used for cultivating the hMSCs. Here, we developed an efficient and robust xeno-free media that will both reduce the manufacturing cost and decrease the regulatory burden associated with bovine serum components found in traditional culture media. We evaluated and compared hMSCs quality parameters in bovine serum-containing and xeno-free media formulations and assessed the cells quality parameters such as cell identity, potency and functionality. hMSC derived in xeno-free media maintain critical functional properties including angiogenic cytokine (FGF, HGF, IL8, TIMP1, TIMP2, and VEGF) secretion, trilineage differentiation, and immunomodulatory potential. In addition, these cells expand rapidly and achieved confluency within 4–5 days of culture without media exchange. The economics of hMSC expansion in this xeno-free media were modeled and compared to other competitive hMSC cell or media systems where it consistently outperformed traditional hMSC systems by more than 8-fold on the critical productivity metric of Million cells per Liter media consumed. The rapid growth and minimal manipulation required with the xeno-free bioprocess media streamlines manufacturing and minimizes the cost of production, making it ideal for use as a commercial platform for various regenerative medicine applications.

Self-cleaning membrane to extend the lifetime of an implanted glucose biosensor.

The lifetime and efficacy of a subcutaneously implanted glucose biosensor could be greatly improved by a self-cleaning membrane capable of periodic physical removal of adhered cells associated with the foreign body reaction. Previously, we reported a thermoresponsive double network nanocomposite (DNNC) membrane composed of poly(N-isopropylacrylamide) (PNIPAAm) and embedded polysiloxane nanoparticles. When the membrane was thermally cycled above and below its volume phase transition temperature (VPTT, ~33-35 °C), the associated deswelling and reswelling, respectively, led to in vitro cell release. Herein, this membrane design was tailored to meet the specific demands of a subcutaneously implanted glucose biosensor, and critical functional properties were assessed. First, N-vinylpyrrolidone (NVP) comonomer increased the VPTT to ~38 °C so that the membrane would be swollen and thus more permeable to glucose in the "off-state" (i.e., no heating) while residing in the subcutaneous tissue (~35 °C). Second, glucose diffusion kinetics though the DNNC membrane was experimentally measured in its deswollen and reswollen states. A cylindrical DNNC membrane with dimensions considered suitable for implantation (1.5 × 5 mm, diameter × length) was used to model the glucose diffusion lag time. In addition, the DNNC cylinder was used to observe dimensional changes associated with deswelling and reswelling. Noncytotoxicity was confirmed and self-cleaning was assessed in vitro in terms of thermally driven cell release to confirm the potential of the DNNC membrane to control biofouling.

Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements

Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.

Critical properties of nanoscale asymmetric ferroelectric tunnel junctions or capacitors

Critical properties, such as the phase transition temperature, critical thickness and Curie–Weiss-type relation, of nanoscale asymmetric ferroelectric tunnel junctions or capacitors are investigated, considering the effects of size, surfaces, asymmetric interfaces and electrodes on the stability and magnitude of the two spontaneous polarization states. Using the modified thermodynamic model and taking into account contributions of the depolarization field, built-in electric field, interface and surface energies to the thermodynamic potential, explicit expressions of the critical properties are derived. For the asymmetric ferroelectric tunnel junction or capacitor, the results illustrate two important behaviors of vanishing critical thickness for the spontaneous polarization and smearing of the phase transitions, respectively. In addition, other critical properties are discussed as functions of the ambient temperature, misfit strain, surface coefficients, work function steps, dielectric constants and screening lengths of electrodes. Owing to the high-sensitivity of the critical properties to structures of asymmetric interfaces and electrodes, the results also suggest that the critical and other functional properties of nanoscale asymmetric ferroelectric tunnel junctions or capacitors can be completely controlled by adjusting the difference between asymmetric interfaces or electrodes.

Improved drug loading/release capacities of commercial contact lenses obtained by supercritical fluid assisted molecular imprinting methods

summary Conventional methods to load drugs into contact lenses are usually inefficient and are mostly limited to high water solubility drugs. This work involves the application of a supercritical fluid assisted molecular imprinting method in order to improve hydro- phobic drug loading into soft contact lenses. The feasibility of preparing improved and more efficient flurbiprofen-loaded Hilafilcon B daily- and monthly-wear contact lenses is demonstrated. Further- more, these processes do not alter critical functional properties of contact lenses and enable the regulation of drug loading by applying various processing cycles.

Supercritical fluid-assisted preparation of imprinted contact lenses for drug delivery

The aim of this work was to develop an innovative supercritical fluid (SCF)-assisted molecular imprinting method to endow commercial soft contact lenses (SCLs) with the ability to load specific drugs and to control their release. This approach seeks to overcome the limitation of the common loading of preformed SCLs by immersion in concentrated drug solutions (only valid for highly water soluble drugs) and of the molecular imprinting methods that require choice of the drug before polymerization and thus to create drug-tailored networks. In particular, we focused on improving the flurbiprofen load/release capacity of daily wear Hilafilcon B commercial SCLs by the use of sequential SCF flurbiprofen impregnation and extraction steps. Supercritical carbon dioxide (scCO2) impregnation assays were performed at 12.0MPa and 40°C, while scCO2 extractions were performed at 20.0MPa and 40°C. Conventional flurbiprofen sorption and drug removal experiments in aqueous solutions were carried out for comparison purposes. SCF-processed SCLs showed a recognition ability and a higher affinity for flurbiprofen in aqueous solution than for the structurally related ibuprofen and dexamethasone, which suggests the creation of molecularly imprinted cavities driven by both physical (swelling/plasticization) and chemical (carbonyl groups in the network with the C–F group in the drug) interactions. Processing with scCO2 did not alter some of the critical functional properties of SCLs (glass transition temperature, transmittance, oxygen permeability, contact angle), enabled the control of drug loaded/released amounts (by the application of several consecutive processing cycles) and permitted the preparation of hydrophobic drug-based therapeutic SCLs in much shorter process times than those using conventional aqueous-based molecular imprinting methods.

Local analysis of the tablet coating process: Impact of operation conditions on film quality

Spray coating is frequently used in the pharmaceutical industry to control the release of the active pharmaceutical ingredient of a tablet or to mask its taste. The uniformity of the coating is of significant importance, as the coating usually has critical functional properties. However, coating uniformity is difficult to predict without significant experimental work, and even advanced particle simulations need to be augmented by CFD models to fully describe the coating uniformity on a single tablet. In this study we analyze the coating process by using detailed computational fluid dynamics (CFD) multiphase spray simulations. The impact and the deposition of droplets on tablets with different shape, as well as the production and evolution of the liquid film on the surface of the tablets are numerically modeled. Spray droplets are simulated with a Discrete Droplets Method (DDM) Euler–Lagrange approach. Models for multi-component evaporation and particle/wall interaction are taken into account. The wall film is treated with a two-dimensional model incorporating submodels for interfacial shear force, film evaporation and heat transfer between film, solid wall and gas phase. Our simulations show how different physical parameters of the coating spray affect the coating process on a single tablet. For example, we analyze for the first time the deposition behavior of the droplets on the tablet. The outcome of our work provides a deeper understanding of the local interaction between the spray and the tablet bed, allowing a step forward in the design, scale-up, optimization and operation of industrial coating devices. Furthermore, it may serve as a basis for the combination with state-of-the-art DEM particle simulation tools.

Decellularized Rhesus Monkey Kidney as a Three-Dimensional Scaffold for Renal Tissue Engineering

The goal of this study was the production of a decellularized kidney scaffold with structural, mechanical, and physiological properties necessary for engineering basic renal structures in vitro. Fetal, infant, juvenile, and adult rhesus monkey kidney sections were treated with either 1% (v/v) sodium dodecyl sulfate or Triton X-100 followed by quantitative and qualitative analysis. Comparison of decellularization agents and incubation temperatures demonstrated sodium dodecyl sulfate at 4 degrees C to be most effective in preserving the native architecture. Hematoxylin and eosin staining confirmed the removal of cellular material, and immunohistochemistry demonstrated preservation of native expression patterns of extracellular matrix proteins, including heparan sulfate proteoglycan, fibronectin, collagen types I and IV, and laminin. Biomechanical testing revealed a decrease in the compressive modulus of decellularized compared to fresh kidneys. Layering of fetal kidney explants on age-matched decellularized kidney scaffolds demonstrated the capacity of the scaffold to support Pax2+/vimentin+ cell attachment and migration to recellularize the scaffold. These findings demonstrate that decellularized kidney sections retain critical structural and functional properties necessary for use as a three-dimensional scaffold and promote cellular repopulation. Further, this study provides the initial steps in developing new regenerative medicine strategies for renal tissue engineering and repair.

The evolutionary pathway from anoxygenic to oxygenic photosynthesis examined by comparison of the properties of photosystem II and bacterial reaction centers

In photosynthetic organisms, such as purple bacteria, cyanobacteria, and plants, light is captured and converted into energy to create energy-rich compounds. The primary process of energy conversion involves the transfer of electrons from an excited donor molecule to a series of electron acceptors in pigment-protein complexes. Two of these complexes, the bacterial reaction center and photosystem II, are evolutionarily related and structurally similar. However, only photosystem II is capable of performing the unique reaction of water oxidation. An understanding of the evolutionary process that lead to the development of oxygenic photosynthesis can be found by comparison of these two complexes. In this review, we summarize how insight is being gained by examination of the differences in critical functional properties of these complexes and by experimental efforts to alter pigment-protein interactions of the bacterial reaction center in order to enable it to perform reactions, such as amino acid and metal oxidation, observable in photosystem II.

BRICHOS - a superfamily of multidomain proteins with diverse functions

BackgroundThe BRICHOS domain has been found in 8 protein families with a wide range of functions and a variety of disease associations, such as respiratory distress syndrome, dementia and cancer. The domain itself is thought to have a chaperone function, and indeed three of the families are associated with amyloid formation, but its structure and many of its functional properties are still unknown.FindingsThe proteins in the BRICHOS superfamily have four regions with distinct properties. We have analysed the BRICHOS proteins focusing on sequence conservation, amino acid residue properties, native disorder and secondary structure predictions. Residue conservation shows large variations between the regions, and the spread of residue conservation between different families can vary greatly within the regions. The secondary structure predictions for the BRICHOS proteins show remarkable coherence even where sequence conservation is low, and there seems to be little native disorder.ConclusionsThe greatly variant rates of conservation indicates different functional constraints among the regions and among the families. We present three previously unknown BRICHOS families; group A, which may be ancestral to the ITM2 families; group B, which is a close relative to the gastrokine families, and group C, which appears to be a truly novel, disjoint BRICHOS family. The C-terminal region of group C has nearly identical sequences in all species ranging from fish to man and is seemingly unique to this family, indicating critical functional or structural properties.

Intrinsic differences between the catalytic properties of the oncogenic NUP214-ABL1 and BCR-ABL1 fusion protein kinases

The NUP214-ABL1 fusion kinase has recently been identified in 6% of patients with T-cell acute lymphoblastic leukemia. In contrast to the more common oncogenic ABL1 fusion BCR-ABL1, NUP214-ABL1 localizes to the nuclear pore complexes and has attenuated transforming properties in hematopoietic cells and in mouse bone marrow transplant models. We have performed a thorough biochemical comparative analysis of NUP214-ABL1 and BCR-ABL1 and show that, despite their common tyrosine kinase domain, the two fusion proteins differ in many critical catalytic properties. NUP214-ABL1 has lower in vitro tyrosine kinase activity, which is in agreement with the absence of phosphorylation on its activation loop. NUP214-ABL1 was more sensitive to imatinib (Glivec) than BCR-ABL1 in vitro and in cells, indicating a different activation state and conformation of the two ABL1 fusion kinases. Using a peptide array, we identified differences in the spectrum and efficiency of substrate peptide phosphorylation and a differential involvement of Src kinases in downstream signaling. These results clearly indicate that different fusion partners of the same kinase can determine not only localization, but also critical functional properties of the enzyme such as inhibitor sensitivity and substrate preference, with subsequent differences in downstream signaling effectors and likely consequences in disease pathogenesis.