Multiple Translocation Research Articles

Multiple proton translocation paths reveal a novel function of Ferritin as a source of protons for cellular metabolism

Multiple proton translocation paths reveal a novel function of Ferritin as a source of protons for cellular metabolism

Brood translocation increases post-release recruitment and promotes population restoration of Centrocercus urophasianus (Greater Sage-Grouse)

Abstract Wildlife managers translocate Centrocercus urophasianus (Greater Sage-Grouse) to augment small populations, but translocated C. urophasianus often fail to reproduce post-release, hampering conservation objectives. We performed 2 C. urophasianus translocations in California and North Dakota from 2017 to 2020 using 2 methods at both sites: an established method of translocating females prior to nesting (i.e., a pre-nesting translocation), and a novel method wherein females were translocated with chicks after successfully hatching nests in source populations (i.e., a brood translocation). We radio-marked and monitored 135 translocated females and 284 source population females and used count data from leks in recipient and source populations to evaluate each method. Using an integrated population model (IPM), we estimated demographic parameters, including recruitment, by females translocated with each method. We estimated the finite rate of change in abundance (λ^) in recipient and source populations given multiple simulated translocation strategies using each method and evaluated them using a cost–benefit metric (i.e., a ratio of the simulated change in abundance at recipient and source sites). We found that recruitment in recipient populations was substantially higher when brood translocations were employed compared to when pre-nesting translocations were employed. Most females translocated pre-nesting forewent reproduction entirely, whereas most females translocated with their broods successfully reared offspring in recipient populations. A per capita comparison revealed that brood translocations resulted in λ^ that were 11–30% greater than pre-nesting translocations, but also revealed that the removal of brood-rearing females from source populations resulted in larger reductions in λ^ when compared with prenesting translocations. However, reductions in λ^ were small and the estimated cost–benefit metrics indicated that translocation of females with broods was a more effective method at spurring growth in recipient populations while minimizing impacts to source populations.

Cytological Assessment of Gamma Induced Multiple Translocation Heterozygote in Sowa (Anethum graveolens L.)

Cytological Assessment of Gamma Induced Multiple Translocation Heterozygote in Sowa (Anethum graveolens L.)

Emerging trends in IRAK-4 kinase research.

The IRAK-4 kinase lies at a critical signaling node that drives cancer cell survival through multiple mechanisms, activation, and translocation of NF-κB mediated inflammatory responses and innate immune signaling through regulation of interferon-α/β receptor (IFNα/β). Inhibition, of IRAK-4, has consequently drawn a lot of attention in recent years to address indications ranging from oncology to autoimmune disorders to neurodegeneration, etc. However, the key stumbling block in targeting IRAK-4 is that despite the inhibition of the kinase activity using an inhibitor the target remains effective, reducing the potential of an inhibitor. This is due to the "scaffolding effect" because of which although regulation of downstream processes by IRAK-4 has been primarily linked with kinase function; however, still, various reports have suggested that IRAK-4 has a non-kinase function in a variety of cell types. This is attributed to the myddosome complex formed by IRAK-4 with myd88, IRAK-2, and IRAK-1 which by itself can cause the activation of downstream effector TRAF6 despite inhibition of the kinase domain of IRAK-4. With this challenge, several groups initiated the development of targeting protein degraders of IRAK-4 using Proteolysis-Targeting Chimeras (PROTACs) technology to completely remove the IRAK-4 from the cellular milieu. In this review, we will capture all these developments and the evolving science around this target.

A Robust FISH Assay to Detect FGFR2 Translocations in Intrahepatic Cholangiocarcinoma Patients.

FGFR fusions retaining the FGFR kinase domain are active kinases that are either overexpressed or constitutively activated throughout diverse cancer types. The presence of FGFR translocations enhances tumor cell proliferation and contributes to significant sensitivity to FGFR kinase inhibitors. FGFR2 as an actionable target in intrahepatic cholangiocarcinoma (iCCA) has been tested in many clinical trials. FISH (fluorescence in situ hybridization) and NGS (next-generation sequence) are well-known tools to investigate the translocations of FGFR with multiple or unknown translocation partners. A rapid and robust FISH assay was developed and validated to detect FGFR2 translocations from FFPE specimens in iCCA. The analytical performance of the FISH assay was evaluated for probe localization, probe sensitivity and specificity, and assay precision. Twenty-five archival FFPE specimens from local iCCA patients were tested for FGFR2 translocations. FISH results were correlated with that of NGS on some samples. Biallelic translocations and a novel FGFR2 translocation involving the partner gene, SHROOM3, t(4;10) (q21;q26), were identified in a local iCCA patient.

Analyzing paramagnetic NMR data on target DNA search by proteins using a discrete-state kinetic model for translocation.

Before reaching their targets, sequence-specific DNA-binding proteins nonspecifically bind to DNA through electrostatic interactions and stochastically change their locations on DNA. Investigations into the dynamics of DNA-scanning by proteins are nontrivial due to the simultaneous presence of multiple translocation mechanisms and many sites for the protein to nonspecifically bind to DNA. Nuclear magnetic resonance (NMR) spectroscopy can provide information about the target DNA search processes at an atomic level. Paramagnetic relaxation enhancement (PRE) is particularly useful to study how the proteins scan DNA in the search process. Previously, relatively simple two-state or three-state exchange models were used to explain PRE data reflecting the target search process. In this work, using more realistic discrete-state stochastic kinetics models embedded into an NMR master equation, we analyzed the PRE data for the HoxD9 homeodomain interacting with DNA. The kinetic models that incorporate sliding, dissociation, association, and intersegment transfer can reproduce the PRE profiles observed at some different ionic strengths. The analysis confirms the previous interpretation of the PRE data and shows that the protein's probability distribution among nonspecific sites is nonuniform during the target DNA search process.

Characterization of novel cell-penetrating peptides derived from the capsid protein of beak and feather disease virus

Beak and feather disease virus (BFDV) is a 17-20 nm icosahedral virus belonging to the Circoviridae family. Psittacine beak and feather disease (PBFD) is caused by BFDV and its common symptoms include abnormal feather, beak, and claw development, as well as immunosuppression in various bird species. In this study, novel cell-penetrating peptides (CPPs) in the capsid protein (Cap) of BFDV were identified through bioinformatic analyses, after which they were experimentally characterized. The cell-penetrating activities of both CPP1 and CPP2 of BFDV were analyzed through flow cytometry and image analysis. The internalization of CPP1 and CPP2 was both dose- and time-dependent but their uptake efficiencies varied depending on the cell type. The cell-penetrating activities of BFDV CPP1 and CPP2 were both superior to that of a typical CPP-TAT originating from the viral protein of human immunodeficiency virus. The cellular uptake of 5 μM CPP1 was close to that of 25 μM TAT, albeit with less cytotoxicity. Using the identified CPPs, the pc-mCheery, pc-Rep, and pc-Cap plasmids were successfully delivered into the target cells for expression. Moreover, both the replication-associated protein with the tag and the Cap protein with the tag could also be successfully delivered into the cells by CPP1 and CPP2. Multiple endocytosis pathways and direct translocation were involved in the cell internalization of CPP1 and CPP2. Furthermore, the delivery of the apoptin gene using CPP1 and CPP2 effectively triggered apoptosis, thus confirming the potential of these CPPs as delivery vehicles. Similarly, green fluorescent protein (GFP) fused with CPP1 or CPP2 at their N-terminus successfully entered the cells. However, the cell internalization efficiency of CPP2-GFP was higher than that of CPP1-GFP. Taken together, our findings demonstrated that both CPP1 and CPP2 of BFDV have promising potential as novel CPPs.

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells. In this study, we have created an unnatural ligand with an unprecedented protein-tag labeling property in living cells and overcome the above-mentioned problem. Of special interest is that some fluorescent probes with the ligand can selectively and efficiently label intracellular proteins without binding to cell-surface proteins, even if the proteins are present on the cell membrane. We also developed a cell-membrane impermeable fluorescent probe that selectively labels cell-surface proteins without labeling of intracellular proteins. These localization-selective properties enabled us to visually discriminate two kinetically distinct glucose transporter 4 (GLUT4) molecules that show different multiple subcellular localization and translocation dynamics in live cells. Taking advantage of the probes, we revealed that N-glycosylation of GLUT4 influences intracellular localization. Furthermore, we were able to visually distinguish active GLUT4 molecules that underwent membrane translocation at least twice within an hour from those that remained intracellularly, discovering previously unrecognized dynamic behaviors of GLUT4. This technology provides not only a valuable tool for study on multiple localization and dynamics of proteins but also important information on diseases caused by protein translocation dysfunction.

Signal sequences encode information for protein folding in the endoplasmic reticulum.

One-third of newly synthesized proteins in mammals are translocated into the endoplasmic reticulum (ER) through the Sec61 translocon. How protein translocation coordinates with chaperone availability in the ER to promote protein folding remains unclear. We find that marginally hydrophobic signal sequences and transmembrane domains cause transient retention at the Sec61 translocon and require the luminal BiP chaperone for efficient protein translocation. Using a substrate-trapping proteomic approach, we identify that nascent proteins bearing marginally hydrophobic signal sequences accumulate on the cytosolic side of the Sec61 translocon. Sec63 is co-translationally recruited to the translocation site and mediates BiP binding to incoming polypeptides. BiP binding not only releases translocationally paused nascent chains but also ensures protein folding in the ER. Increasing hydrophobicity of signal sequences bypasses Sec63/BiP-dependent translocation, but translocated proteins are prone to misfold and aggregate in the ER under limited BiP availability. Thus, the signal sequence-guided protein folding may explain why signal sequences are diverse and use multiple protein translocation pathways.

Combination venetoclax and selinexor effective in relapsed refractory multiple myeloma with translocation t(11;14)

Patients with multiple myeloma-bearing translocation t(11;14) have recently been shown to benefit from the apoptosis-inducing drug venetoclax; however, the drug lacks FDA approval in multiple myeloma thus far due to a potential safety signal in the overall patient population. Selinexor is an inhibitor of nuclear export that is FDA-approved for patients with multiple myeloma refractory to multiple lines of therapy. Here, we report that in four patients with multiple myeloma with t(11;14), the concomitant administration of venetoclax and selinexor was safe and associated with disease response. Moreover, the combination was synergistic in t(11;14) multiple myeloma cell lines and caused decreased levels of Cyclin D1 (which is overexpressed due to the CCND1-IGH fusion) when given in combination as compared to single agents. These data suggest that the combination of venetoclax and selinexor is effective and t(11;14) may serve as a therapeutic marker for response and target for future clinical trials.

Elucidating the dynamics of polymer transport through nanopores using asymmetric salt concentrations

While notable progress has been made in recent years both experimentally and theoretically in understanding the highly complex dynamics of polymer capture and transport through nanopores, there remains significant disagreement between experimental observation and theoretical prediction that needs to be resolved. Asymmetric salt concentrations, where the concentrations of ions on each side of the membrane are different, can be used to enhance capture rates and prolong translocation times of electrophoretically driven polymers translocating through a nanopore from the low salt concentration reservoir, which are both attractive features for single-molecule analysis. However, since asymmetric salt concentrations affect the electrophoretic pull inside and outside the pore differently, it also offers a useful control parameter to elucidate the otherwise inseparable physics of the capture and translocation process. In this work, we attempt to paint a complete picture of the dynamics of polymer capture and translocation in both symmetric and asymmetric salt concentration conditions by reporting the dependence of multiple translocation metrics on voltage, polymer length, and salt concentration gradient. Using asymmetric salt concentration conditions, we experimentally observe the predictions of tension propagation theory, and infer the significant impact of the electric field outside the pore in capturing polymers and in altering polymer conformations prior to translocation.

Translocation of mitochondrial DNA into the nuclear genome blurs phylogeographic and conservation genetic studies in seabirds.

Mitochondrial DNA (mtDNA) translocated into the nuclear genome (numt), when co-analysed with genuine mtDNA, could plague phylogeographic studies. To evaluate numt-related biases in population genetics parameters in birds, which are prone to accumulating numts, we targeted the mitochondrial mt-cytb gene. We looked at 13 populations of Audubon's shearwater (Puffinus lherminieri), including five mitochondrial lineages. mt-cytb homologue and paralogue (numt) sequences were determined by Sanger sequencing with and without prior exonuclease digestion of nuclear DNA. Numts formed monophyletic clades corresponding to three of the five mitochondrial lineages tested (the remaining two forming a paraphyletic group). Nineteen percent of numt alleles fell outside of their expected mitochondrial clade, a pattern consistent with multiple translocation events, incomplete lineage sorting (ILS), and/or introgression. When co-analysing mt-cytb paralogues and homologues, excluding individuals with ambiguities underestimates genetic diversity (4%) and differentiation (11%) among least-sampled populations. Removing ambiguous sites drops the proportion of inter-lineage genetic variance by 63%. While co-analysing numts with mitochondrial sequences can lead to severe bias and information loss in bird phylogeographic studies, the separate analysis of genuine mitochondrial loci and their nuclear paralogues can shed light on numt molecular evolution, as well as evolutionary processes such as ILS and introgression.

AAA+ proteins: one motor, multiple ways to work.

Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined. In the cases where translocation mechanisms have been determined, a two-residue translocation step-size has not been resolved. In this review, we explore Hsp104, ClpB, ClpA and ClpX as examples to review the experimental methods that have been used to examine, in solution, the translocation mechanisms employed by AAA+ motor proteins. We then ask whether AAA+ motors sharing similar structural features can have different translocation mechanisms. Finally, we discuss whether a single AAA+ motor can adopt multiple translocation mechanisms that are responsive to different challenges imposed by the substrate or the environment. We suggest that AAA+ motors adopt more than one translocation mechanism and are tuned to switch to the most energetically efficient mechanism when constraints are applied.

Flavors of Non-Random Meiotic Segregation of Autosomes and Sex Chromosomes

Segregation of chromosomes is a multistep process occurring both at mitosis and meiosis to ensure that daughter cells receive a complete set of genetic information. Critical components in the chromosome segregation include centromeres, kinetochores, components of sister chromatid and homologous chromosomes cohesion, microtubule organizing centres, and spindles. Based on the cytological work in the grasshopper Brachystola, it has been accepted for decades that segregation of homologs at meiosis is fundamentally random. This ensures that alleles on chromosomes have equal chance to be transmitted to progeny. At the same time mechanisms of meiotic drive and an increasing number of other examples of non-random segregation of autosomes and sex chromosomes provide insights into the underlying mechanisms of chromosome segregation but also question the textbook dogma of random chromosome segregation. Recent advances provide a better understanding of meiotic drive as a prominent force where cellular and chromosomal changes allow autosomes to bias their segregation. Less understood are mechanisms explaining observations that autosomal heteromorphism may cause biased segregation and regulate alternating segregation of multiple sex chromosome systems or translocation heterozygotes as an extreme case of non-random segregation. We speculate that molecular and cytological mechanisms of non-random segregation might be common in these cases and that there might be a continuous transition between random and non-random segregation which may play a role in the evolution of sexually antagonistic genes and sex chromosome evolution.

High Levels of Genetic Divergence Detected in Sacramento Perch Suggests Two Divergent Translocation Sources

AbstractTranslocation has been used to conserve imperiled fishes and create new fisheries. One species for which translocation has played a significant role is the Sacramento Perch Archoplites interruptus. Extirpated from its native range, the Sacramento Perch has been introduced throughout California and Nevada through multiple translocation events, though historical records are incomplete. Recent assessments of eight previously uncharacterized Sacramento Perch populations have prompted reevaluation of range‐wide population structure to inform a genetic management plan for long‐term resiliency of this species. We examined Sacramento Perch genetic diversity and population structure across the current range of the species using 12 microsatellite markers. We analyzed samples from the eight uncharacterized populations and seven populations previously studied by Schwartz and May (2008). Bayesian clustering supported two distinct clusters of Sacramento Perch herein designated as A and B. Within these two clusters we detected hierarchical substructure, likely due to genetic drift after population founding. Genetic differentiation among populations within the same cluster was relatively low (FST = 0.023–0.176), while differentiation among populations from different clusters was higher (FST = 0.190–0.320). The existence of two strongly divergent genetic clusters in Sacramento Perch suggests two distinct translocation sources, and we recommend that these clusters be treated as genetic management units (GMUs). The B GMU populations had fairly low levels of genetic diversity relative to the A GMU populations. All populations showed evidence of past bottlenecks, and most had effective population sizes placing them at risk for inbreeding depression. Human‐facilitated gene flow is recommended to prevent further genetic diversity loss. Due to uncertainty surrounding Sacramento Perch translocation history and strong levels of divergence between the two GMUs, translocations should be facilitated only between populations within the same GMU.

Gamma irradiation induced multiple chromosome interchanges in Hordeum vulgare L. (Poaceae): meiotic characterization and their implications on pollen fertility

Barley (Hordeum vulgare L.) is an important cereal crop with 2n = 2x = 14 chromosomes. Gamma irradiation treatments to barley seeds successfully induced four multiple translocation heterozygotes (T-1, T-2 and T-3 from 25 kR, and T-4 from 20 kR) in M1 generation. In meiosis I, microsporocytes of T-1, T-2 and T-3 heterozygotes showed 2 rings/chains of four chromosomes, whereas T-4 displayed a ring/chain of six chromosomes. In this study, quadrivalents showed preponderance of ring configurations (ranged from 63.27 to 69.42%), while majority of hexavalents revealed chain configurations (63.41%). Due to meiotic instability, hexavalents sometimes gets dissociated into pentavalents, quadrivalents, trivalents along with univalents and bivalents. Assessment of orientation pattern of interchange complexes in the heterozygotes indicated alternate disjunction to be more frequent than adjacent type of disjunction. Presumably, involvement of more number of non-homologous chromosomes in translocation heterozygotes resulted in increased chromosome irregularities during meiotic segregation which ultimately led to much higher pollen sterility and concomitant decrease in growth and seed formation. In addition, pollen sterility (ranged from 47.97 to 58.09%) showed positive and significant correlation (r2 = 0.6222) with adjacent segregation of multiples (37.84–44.61%) in translocation heterozygotes.

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

Genetic outcomes of translocation of bighorn sheep in Arizona

ABSTRACTTranslocation is an important management tool that has been used for >50 years in Arizona, USA, to increase bighorn sheep (Ovis canadensis) population densities and to restore herds to suitable habitat throughout their historical range. Yet, translocation can also alter the underlying genetic diversity and spatial structure of managed wildlife species in beneficial or detrimental ways. To evaluate the long‐term effect of translocation actions on bighorn sheep, we characterized statewide genetic structure and diversity using microsatellite and mitochondrial DNA data in 16 indigenous and translocated (supplemented or reintroduced) Arizona populations sampled between 2005 and 2012. Populations that were recipients of translocated animals showed no reduction in genetic diversity with allelic richness and heterozygosity estimates equivalent to, and in some cases greater than, indigenous source populations. The indigenous population occupying the Silver Bell Mountains population displayed the lowest indices of genetic diversity but shared mitochondrial DNA haplotypes with the Mohawk Mountains, Sierra Pinta, and Cabeza Prieta populations, indicating past connectivity and potential opportunities for genetic management if warranted. Bayesian clustering on genetic similarity and genetic divergence estimates corroborated previous work differentiating Rocky Mountain bighorn sheep (O. c. canadensis) and 2 desert lineages corresponding with Nelson's (O. c. nelsoni) and Mexican desert bighorn sheep (O. c. mexicana). In northern Arizona, assignment tests confirmed the presence of 2 indigenous metapopulations of Nelson's desert bighorn sheep in the Black Mountains and Grand Canyon and indicated that gene flow from the Grand Canyon population has likely played a role in maintaining genetic diversity and mitigating founder effects among multiple translocated populations in the area. In southern Arizona, we detected genetic structure consistent with 2 metapopulations of Mexican desert bighorn sheep representing a departure from current management practices that consider this lineage to be a single genetic unit. Several lines of genetic evidence presented in this study suggest that the Bill Williams River area is the contemporary contact zone for the 2 desert lineages; however, the degree to which translocation has enhanced introgression is unknown. Despite relative isolation from other herds, the translocated Rocky Mountain bighorn sheep population in eastern Arizona had high levels of allelic richness and heterozygosity and a negative inbreeding coefficient, conceivably as a result of multiple translocation events from sources in Colorado and New Mexico, USA. Although translocation management has successfully contributed to the reestablishment of bighorn sheep populations in Arizona without diminishing genetic diversity, future translocation should proceed with caution to preserve the genetic integrity and potential local adaptation within the Nelson's and Mexican desert bighorn sheep metapopulations identified in this study. © 2019 The Wildlife Society.

The genetic legacy of 50 years of desert bighorn sheep translocations.

Conservation biologists have increasingly used translocations to mitigate population declines and restore locally extirpated populations. Genetic data can guide the selection of source populations for translocations and help evaluate restoration success. Bighorn sheep (Ovis canadensis) are a managed big game species that suffered widespread population extirpations across western North America throughout the early 1900s. Subsequent translocation programs have successfully re‐established many formally extirpated bighorn herds, but most of these programs pre‐date genetically informed management practices. The state of Nevada presents a particularly well‐documented case of decline followed by restoration of extirpated herds. Desert bighorn sheep (O. c. nelsoni) populations declined to less than 3,000 individuals restricted to remnant herds in the Mojave Desert and a few locations in the Great Basin Desert. Beginning in 1968, the Nevada Department of Wildlife translocated ~2,000 individuals from remnant populations to restore previously extirpated areas, possibly establishing herds with mixed ancestries. Here, we examined genetic diversity and structure among remnant herds and the genetic consequences of translocation from these herds using a genotyping‐by‐sequencing approach to genotype 17,095 loci in 303 desert bighorn sheep. We found a signal of population genetic structure among remnant Mojave Desert populations, even across geographically proximate mountain ranges. Further, we found evidence of a genetically distinct, potential relict herd from a previously hypothesized Great Basin lineage of desert bighorn sheep. The genetic structure of source herds was clearly reflected in translocated populations. In most cases, herds retained genetic evidence of multiple translocation events and subsequent admixture when founded from multiple remnant source herds. Our results add to a growing literature on how population genomic data can be used to guide and monitor restoration programs.

Allosteric regulation alters carrier domain translocation in pyruvate carboxylase

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The reaction occurs in two separate catalytic domains, coupled by the long-range translocation of a biotinylated carrier domain (BCCP). Here, we use a series of hybrid PC enzymes to examine multiple BCCP translocation pathways in PC. These studies reveal that the BCCP domain of PC adopts a wide range of translocation pathways during catalysis. Furthermore, the allosteric activator, acetyl CoA, promotes one specific intermolecular carrier domain translocation pathway. These results provide a basis for the ordered thermodynamic state and the enhanced carboxyl group transfer efficiency in the presence of acetyl CoA, and reveal that the allosteric effector regulates enzyme activity by altering carrier domain movement. Given the similarities with enzymes involved in the modular synthesis of natural products, the allosteric regulation of carrier domain movements in PC is likely to be broadly applicable to multiple important enzyme systems.