In particular, the researchers found a critical gene in heparin biosynthesis: ZNF263 (zinc-finger protein 263). The researchers believe this gene regulator is a key discovery on the way to industrial heparin production. The idea would be to control this regulator in industrial cell lines using genetic engineering, paving the way for safe industrial production of heparin in well-controlled cell culture.

Heparin is currently produced by extracting the drug from pig intestines, which is a concern for safety, sustainability, and security reasons. Millions of pigs are needed each year to meet our needs, and most manufacturing is done outside the USA. Furthermore, ten years ago, contaminants from the pig preparations led to dozens of deaths. Thus, there is a need to develop sustainable recombinant production. The work in PNAS provides new insights on exactly how cells control synthesis of heparin.

Heparin regulation

Heparin is a special subtype of a more general class of carbohydrates, called heparan sulfates, that are produced by a wide range of cells, both in the human body, as well as in cell culture. Yet, heparin is exclusively produced in a special type of blood cells called mast cells. To this day, heparin cannot be successfully produced in cell culture.

Researchers at UC San Diego reasoned that heparin synthesis must be under the control of certain gene regulators (called transcription factors), whose tissue-specific occurrence might give mast cells the unique ability to produce heparin.

Since regulators for heparin were not known, a research team led by UC San Diego professors Jeffrey Esko and Nathan Lewis used bioinformatic software to scan the genes encoding enzymes involved in heparin production and specifically look for sequence elements that could represent binding sites for transcription factors. The existence of such a binding site could indicate that the respective gene is regulated by a corresponding gene regulator protein, i.e. a transcription factor.

“One DNA sequence that stood out the most is preferred by a transcription factor called ZNF263 (zinc-finger protein 263),” explains UC San Diego professor Nathan E. Lewis, who holds appointments in the UC San Diego School of Medicine’s Department of Pediatrics and in the UC San Diego Jacobs School of Engineering’s Department of Bioengineering.

“While some research has been done on this gene regulator, this is the first major regulator involved in heparin synthesis,” said Lewis. He is also Co-Director of the CHO Systems Biology Center at the UC San Diego Jacobs School of Engineering.

Using the gene-editing technology, CRISPR/Cas9, the UC San Diego researchers mutated ZNF263 in a human cell line that normally does not produce heparin. They found that the heparan sulfate that this cell line would normally produce was now chemically altered and showed a reactivity that was closer to heparin.

Experiments further showed that ZNF263 represses key genes involved in heparin production. Interestingly, analysis of gene expression data from human white blood cells showed suppression of ZNF263 in mast cells (which produce heparin in vivo) and basophils, which are related to mast cells. The researchers report that ZNF263 appears to be an active repressor of heparin biosynthesis throughout most cell types, and mast cells are enabled to produce heparin because ZNF263 is suppressed in these cells.

This finding could have important relevance in biotechnology. Cell lines used in industry (such as CHO cells that normally are unable to produce heparin) could be genetically modified to inactivate ZNF263 which could enable them to produce heparin, like mast cells do.

Philipp Spahn, a project scientist in Nathan Lewis’ lab in the Departments of Pediatrics and Bioengineering at UC San Diego, described further directions the team is pursuing: “Our bioinformatic analysis revealed several additional potential gene regulators which can also contribute to heparin production and are now exciting objects of further study.”

The team also showed that PARP inhibitor compounds can be “tuned” in the opposite way so that they inhibit PARP-1 enzymes without killing cells, thus potentially making this class of drugs more useful for treating heart disease and other non-cancer conditions where inhibiting PARP-1 is the goal.

“We can now use this new understanding of how PARP inhibitors work to design compounds that are better tailored for specific conditions such as cancers vs. heart disease,” said senior author Ben Black, PhD, the Eldridge Reeves Johnson Foundation Professor of Biochemistry and Biophysics and co-director of the Penn Center for Genome Integrity at the Perelman School of Medicine at the University of Pennsylvania.

PARP-1 is a DNA-repair enzyme that becomes particularly important for cell survival when other major DNA-repair factors such as BRCA proteins are missing. PARP inhibitors, of which four have been approved so far by the U.S. Food and Drug Administration, are thus considered especially promising as treatments for cancers driven by BRCA gene mutations. Tumor cells driven by BRCA mutations typically lack any BRCA-based DNA-repair capability, and their exposure to a PARP inhibitor further degrades their ability to fix DNA breaks, making the cells much more likely to die. PARP inhibitors have been shown to improve cancer patient outcomes when used alone or in combination with therapies such as chemo and radiation that induce DNA damage.

Despite promising clinical results for several PARP-1 inhibitors, a mystery has surrounded this drug class. All of these compounds bind tightly to the active site of the PARP-1 enzyme and thus all should inhibit the enzyme very effectively — yet only some of these compounds show potency at killing tumor cells, while others don’t.

In recent years, scientists have found increasing evidence that PARP inhibitors kill cancer cells not just by inhibiting PARP-1 activity, but also by somehow trapping PARP-1 enzymes on the DNA breaks they are attempting to fix. Keeping PARP-1 enzymes stuck to a cell’s DNA effectively kills the cell when it attempts to divide — and cancer cells divide relatively frequently. The idea that PARP inhibitors vary in their abilities to “trap” PARP-1 enzymes on DNA has emerged as a potential explanation for these compounds’ variable cancer-killing effects.

In the new study, Black and his team used sophisticated and sensitive techniques to show that PARP inhibitors bind to the PARP-1 enzyme in ways that weaken or strengthen the enzyme’s attachment to DNA breaks. These techniques, which include atomic level structural determination and probing the dynamics of the amide protons of the backbone of PARP-1, go beyond what previous research has done to understand how PARP inhibitors actually work. Veliparib, a PARP-1 inhibitor that has puzzled pharmaceutical scientists with its failure in recent clinical trials against breast and lung cancers, turns out to weaken PARP-1’s grip on DNA, which in principle makes it easier for a veliparib-treated tumor cell to survive.

The team showed, however, that they could chemically modify veliparib to greatly boost its ability to trap PARP-1 on DNA. This in turn greatly boosted its cancer-cell-killing effects in experiments with different types of cancer cell.

“It was clear that the increased potency of the new compound relative to unmodified veliparib is due to its increased ability to keep PARP-1 bound to DNA breaks,” Black said.

The work greatly clarifies the complex interaction between PARP-1 inhibitors and the PARP-1 enzyme, and shows how variations in this interaction correspond to different cell-killing effects. In principle, Black noted, this improved understanding can now be used not only to design more potent anticancer PARP-1 inhibitors, but also to design PARP-1 inhibitors that don’t kill cells by trapping PARP-1 on DNA.

The latter could be useful in treating inflammation, heart disease, stroke, and other conditions that have been shown to involve excessive PARP-1 activity, potentially including COVID-19.

The work was supported by the Canadian Institute of Health Research (BMA342854), the US National Institutes of Health (R35GM130302, F32GM128265), a Basser Center for BRCA Award, the Department of Pharmaceutical Sciences and seed grant program of St. John’s University (579-1110-6709), the Breast Cancer Now charity (CTR-Q4-Y2), and Cancer Research UK (CRUK/A14276).

“Identifying and characterizing such topological phases in experiments is a great challenge,” say Benoît Vermersch, Jinlong Yu and Andreas Elben from the Center for Quantum Physics at the University of Innsbruck and the Institute for Quantum Optics and Quantum Information of the Austrian Academy of Sciences. “Topological phases cannot be identified by local measurements because of their special properties. We are therefore developing new measurement protocols that will enable experimental physicists to characterize these states in the laboratory.” In recent years this has already been achieved for non-interacting systems. However, for interacting systems, which in the future could also be used as topological quantum computers, this has not been possible so far.

With random measurements to a definite result

In Science Advances, the physicists of Peter Zoller’s research group now propose measurement protocols that enable the measurement of so-called topological invariants. These mathematical expressions describe common properties of topological spaces and make it possible to fully identify interacting topological states with global symmetry in one-dimensional, bosonic systems. “The idea of our method is to first prepare such a topological state in a quantum simulator. Now so-called random measurements are performed, and topological invariants are extracted from statistical correlations of these random measurements,” explains Andreas Elben. The specific feature of this method is that although the topological invariants are highly complex, non-local correlation functions, they can still be extracted from statistical correlations of simple, local random measurements. As with a method recently presented by the research group for comparing quantum states in computers or simulators, such random measurements are possible in experiments today. “Our protocols for measuring the topological invariants can therefore be directly applied in the existing experimental platforms,” says Benoît Vermersch.

Story Source:

Materials provided by University of Innsbruck. Note: Content may be edited for style and length.

Asgard archaea are relatively new to science, but they are ancient and important to us in more ways than one. They are single-celled organisms and were originally found at the bottom of the ocean. Asgard archaea are what are known as a prokaryote, they do not have a cell nucleus, yet despite this, they are genetically close to single-celled organisms called eukaryotes which do contain a cell nucleus. They are like a modern analogue of an ancient common ancestor.

The race is on to investigate these small but significant organisms. Associate Professor Keiichi Inoue from the Institute for Solid State Physics at the University of Tokyo, Professor Hideki Kandori from Nagoya Institute of Technology and their team chose to study a feature of Asgard archaea that although not unique to them, is especially interesting in their case, and that is light-sensitive or photoreceptive proteins called rhodopsins. The organisms live at the bottom of oceans and lakes so it’s surprising they need any kind of sensitivity to light.

“We explored the molecular function of special rhodopsins in Asgard archaea called schizorhodopsins and found that they acted as light-activated microscopic pumps,” explained Inoue. “Schizorhodopsin uses sunlight energy to take up a proton into the cell along a pathway inside the protein. Many prokaryotes such as bacteria and other archaea use rhodopsins to pump protons out, but we find this newly characterized form in Asgard archaea particularly interesting.”

As the scale this function occurs on is nanoscopic, sophisticated measurement techniques with high sensitivity and high temporal resolution were required. Inoue, Kandori and their team used a method called laser flash photolysis which uses pulsed laser light to stimulate reactions. Color change in the protein affected by laser light was monitored by sensitive sensors. These detected the presence and nature of the short-lived activation of schizorhodopsin.

“These findings will help us better understand proton and other ion transport mechanisms. In addition, schizorhodopsin could be made into a useful molecular tool for researchers,” commented Inoue. “For example in optogenetics, which is a new methodology to control various cellular phenomena with light. Schizorhodopsins could also be used to control the pH inside cells or microorganisms with light, as pH can be altered by changing the proton concentration.”

Story Source:

Materials provided by University of Tokyo. Note: Content may be edited for style and length.