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Will new proteins incorporating new amino acids trigger an immune response?

Will new proteins incorporating new amino acids trigger an immune response?


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This article reported that scientists have succeeded in adding two new bases to the quartet of A, C, G and T, resulting in non-canonical amino acid. Additionally, the bacteria in which this was done were able to produce new proteins using the newly added bases. The article quotes the scientists as saying that the extra amino acids “might become building blocks for new drugs and novel materials”.

My question is that if new proteins are made from amino acids that don't naturally occur, won't the body reject them?


The body's mechanism for detecting foreign object has a built-in failsafe that minimizes the likelihood of misinterpreting a self-derived antigen as foreign.

Immunologic tolerance (unresponsiveness) normally prevents reactions against self-antigens; if immunologic tolerance is broken, autoimmune reactions may occur. Much of the development of tolerance occurs in the thymus by the elimination (clonal deletion) or inactivation (clonal anergy) of self-reactive clones of T cells. Other mechanisms of tolerance occur extrathymically and include activation of antigen-specific T suppressor cells and clonal deletion, which results in the elimination of self-reactive B cells or T cells, and clonal anergy.

https://www.ncbi.nlm.nih.gov/books/NBK7795/

Basically, before a T cell starts looking for potential antigens, it generally first spends some time in the thymus being tested against antigens which are native to the body. If it responds to such an antigen, the T cell will either be destroyed or deactivated.

Having unnatural amino acids as part of this process is unlikely to change its efficacy. T cells which would target peptides with these unnatural amino acids will still be checked against the body's cells before being sent out to do their work; the mechanism would work the same way. I suspect that the rate of autoimmune diseases would be unchanged compared to normal organisms.


Introduction

This question has two components. The first is the near rhetorical question whether proteins with unnatural amino acids will be considered by the immune system as 'non-self'. Unless, by chance, the unnatural amino acid resembles some normal macromolecular component, the answer is obviously that it will be regarded as 'non-self'.

The second question is, to my mind, much more interesting, but does not appear to be considered (or has been taken for granted) in the answer by @Astrolamb. This is whether the immune response would cause such artificial proteins to be eliminated, if administered therapeutically. It is important, at this juncture, to emphasize that proteins containing unnatural amino acids as the result of expanding the genetic code do not, in principle, raise any problems of immune 'rejection' that have not already been seen in 'unnatural' proteins containing normal amino acids. Any immune response should therefore be regarded as being to 'non-self protein' and rather than to 'non-self amino acid'.

As there have been many such studies with the latter proteins (and with proteins containing unnatural amino acids introduced by chemical modification) there are extensive studies on which to base an answer. An immunologist would be a better person to do this, but in the absence of any “immune response” I will attempt an answer from my perusal of the literature, leaning particularly on:

K. Wals and H. Ovaa “Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins” Front. Chem 2, 1-12 (2014)

and

B. Leader et al. “Protein therapeutics: a summary and pharmacological classification” Nature Reviews Drug Discovery 7, 21-39 (2008)

I welcome comments suggesting improvements or corrections.

Summary

The question asks:

“if new proteins are made from amino acids that don't naturally occur, won't the body reject them?”

which I take to mean:

“if new proteins are made from amino acids that don't naturally occur, won't they induce an immune response which will render them ineffective?”

The brief answer to this is:

  1. It depends - the immunogenicity of foreign proteins varies.
  2. Even if an immune response is elicited this may not necessarily render such proteins ineffective.
  3. In some cases the therapeutic intention is actually to provoke an immune response - to make a weakly immunogenic protein more so.

Foreign proteins vary in immunogenicity

The crux of the answer to this question lies in the following:

The fact that a protein used for therapeutic purposes differs from those in a human subject does not in itself mean that it will invoke an immune response.

Let us consider two examples to illustrate this.

The first is the case of porcine insulin, which has been administered to patients for many years, but has one amino acid different from human insulin. Regarding such non-human insulin this review states that “severe immunological complications occur rarely, and less severe events affect a small minority of patients”.

The second example is taken from more recent work by D-A. Silva et al. in designing artificial proteins. (See also Nature News & Views summary). They designed mimics of the cytokines, interleukin-2 and interleukin-5, that had extensive differences in helix organization and amino acid sequence from the native proteins. However they reported that “Immunogenicity against the de novo designed proteins appears to be low”.

It turns out that a wide variety of factors affect the immunogenicity of foreign macromolecules, so that even if a therapeutic protein elicits an immune response it may be weak enough to allow the therapeutic action to occur. In the work just cited, Silva et al. suggest that the reason for the low immunogenicity might be “the small size and high stability” of the proteins on the basis of previous work they had performed with 20,000 mini-proteins.

Admittedly, in some cases a strong immune response will occur and can obviously be a problem, so that measures need to be taken to reduce the immunogenicity. This is widely recognized and discussed in the following paper:

V. Brinks et al. “Immunogenicity of Therapeutic Proteins: The Use of Animal Models” Pharm Res (2011) 28:2379-2385

Sometimes chemical modification of proteins can actually reduce their immunogenicity, as is the case for polyethylene glycol in relation to interferon (B. Leader et al. review, cited above).

Use of therapeutic proteins to provoke an immune response

One use envisaged for modified proteins is in cancer immunotherapy. In brief, the objective is to try to make the body mount an immune response against cancer cells, which may exhibit 'foreign' tumour antigens. However, in many cases these differences from normal cells are not sufficient to provoke an adequate immune response. It is has been shown that modifying retinol binding protein by introducing an unnatural amino acid can help overcome the self-tolerance to tumours in mice, suggesting a possible use in cancer immunotherapy (J. Grünewald et al. “Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids” PNAS (2009) 106, 4337-4342).

Conclusion

Proteins containing unnatural amino acids as the result of expanding the genetic code do not, in principle, raise any problems of immune 'rejection' that have not already been seen in 'unnatural' proteins containing normal amino acids or chemically modified amino acids. The problem of potential immunogenicity is widely recognized and has been extensively studied by those wishing to develop the considerable therapeutic potential of all such proteins.


Could an extremophile hold the secret to treatment of devastating injuries?

An illustration of a tardigrade, which is capable of withstanding dehydration and cosmic radiation. Credit: iStock/Eraxion

Water bear. Moss piglet. Tardigrade

The gentle teddy-bear features of this polyonymic animal belie its hardy nature.

Capable of withstanding dehydration and cosmic radiation and surviving temperatures as low as -450 degrees F and as high as 300 degrees F, this eight-limbed microscopic creature holds the key to one of biology's greatest secrets—extreme survival.

Tardigrades have captivated the imagination of astrobiologists—several of the clan's members have traveled to space as part of research experiments—and tantalized the fantasies of sci-fi fans as a giant alien creature in Star Trek: Discovery.

Now scientists at Harvard Medical School (HMS) are wondering: Can the physiology of this extremophile yield insights that can be applied to humans?

Inspired by nature, optimized in the lab

The HMS group, working with colleagues at the University of Washington, Seattle, and MIT, are hoping to answer this very question in an ambitious new project aimed at deciphering the structure and function of several tardigrade proteins suspected to play a role in the organism's resilience, and then use these proteins as the basis for human therapies that halt tissue damage and prevent cell death.

The team's goal is to engineer an optimized version of these proteins and use them to slow down metabolic activity in injured cells—the biological equivalent of hitting the pause button on cellular processes, including damage-causing inflammation, infection and, ultimately, cell demise.

The team's ultimate goal is to develop a protein-based therapy that can halt tissue damage in traumatic injuries, heart attacks, strokes and sepsis, among other conditions.

"It really started out as a wacky, high-risk idea," said Pamela Silver, the project's principal co-investigator and professor of systems biology in the Blavatnik Institute at HMS and member of the Harvard Wyss Institute at Harvard.

In the spring of 2018, Silver came across a grant challenge posted by the U.S. military seeking novel solutions to stabilizing traumatic injuries in combat zones. She knew just whose brain to pick.

Roger Chang, a bioinformatician and molecular biologist in Silver's lab, has long been fascinated with protein resistance, studying, among other things, the role of proteins in thermal stress and shielding bacterial cells from gamma radiation. Not long ago, Chang had seen a study showing that when tardigrade proteins are introduced in E.coli and yeast, they render the organisms unusually tolerant to desiccation.

What if these processes could be replicated in human cells, Chang wondered. The wild-type tardigrade protein showed only modest protective effect in E.coli and yeast, but what if these proteins could be made more potent in the lab, Chang asked.

"I started conceiving how we could improve upon nature's 'raw' materials and functionalize them for human use," Chang said.

The beauty of disorder

What allows tardigrades to survive in conditions that would kill most other organisms? The precise mechanisms behind their ability to undergo self-preserving cryptobiosis remain poorly understood. However, evidence suggests that when it undergoes this process, it deploys a series of biochemicals—including proteins and sugar molecules—that shield nucleic acids and proteins inside cells from damage.

The proteins in question belong to a class known as intrinsically disordered proteins (IDPs). IDPs' most striking feature—which gives the class its name—is the lack of neat, orderly structure that is easily visualized in 3-D imaging studies.

Tardigrades are, by no means, the only animals exhibiting the presence of IDPs. These proteins are nearly ubiquitous across species, including humans. Nor are tardigrades the only organisms capable of surviving extreme stress. Other known extremophiles include certain nematodes, brine shrimp and the poetically named resurrection plant, which can survive dehydration for years and bloom back into life within mere hours of being watered.

Biologists have been aware of the existence of disordered proteins for decades but long dismissed them as inconsequential. IDPs are so poorly understood that there is still no clear consensus on what constitutes one. In fact, for the most part, their functions remain unknown. What scientists do know is that not all disordered proteins are capable of slowing down cellular activity. Mapping out their structure remains a fundamental problem—a challenge that leaves scientists wondering which among these proteins are truly disordered and which ones are simply not amenable to current structure-mapping techniques.

Designing any protein is a formidable challenge—not unlike designing a car. Designing a disordered protein is akin to creating the blueprint for an aircraft.

Silver and Chang knew it was time to call on Debora Marks, a mathematician and computational biologist in the Blavatnik Institute at HMS with a keen interest in IDPs and expertise in predictive computer modeling.

It would be a daunting task. Yet, combining Silver's expertise in synthetic biology with Marks' prowess in machine learning and computation would put this within the realm of the possible.

Computer-designed proteins

As recently as 10 years ago, synthetic biologists would approach protein development by brute force—trial-and-error design followed by testing in a long, seemingly interminable, string of experiments to arrive at the right recipe. Today, advances in computational biology and machine learning are making the process more focused and efficient.

A protein is a string of amino acids, the sequence of which determines the protein's shape. The shape, in turn, dictates the protein's function and role: what it can and cannot do. The number of possible amino acid combinations is infinite so how does one pick the right sequence to make a protein that performs a predetermined set of biological tasks?

Marks, the projects other principal co-investigator, will spearhead the computational design of synthetic IDPs suitable for use in human cells. What this entails, Marks explains, is discovering which features of amino acid sequences in a protein would allow it to slow a cell's biological functions and do so reversibly. The task would require the use of probabilistic computer modelling to build a protein that is both safe and functional in humans. A critical prerequisite for use in humans would be ensuring that the protein can bypass the body's immune defenses. In other words, the candidate protein should not trigger an antibody response in human tissues and cause an attack by the host's immune system. To optimize the protein's design features, Marks will collaborate with David Baker at the University of Washington to predict how the protein would interact with tens of thousands of cellular components when introduced inside human cells.

Rather than testing infinite possibilities, this predictive design is targeted, Marks said. It is an informed pretesting of a finite, and hopefully, small number of possible amino acid combinations.

"This is not a black-box approach where we throw in every possible combination and put in a load of features and see what sticks," Marks said. "It is a form of unsupervised machine learning that doesn't presume an outcome. The universe of possible protein sequences is infinite, so we want to be directed and targeted."

The team will start out by testing the candidate proteins in human cells derived from various tissues—muscle, vascular, cardiac, nerve cells, and so forth. Then they will test the protein in human organoids and, finally in animals. It would be possible to precision-target the protein to specific cell, tissues and organ types, Silver said.

"We've studied protein targeting for years, and we have a trick we've developed on how to home in a protein on specific targets but not others," Silver said.

How the damage-halting protein will be delivered inside cells will be one of the fundamental challenges in this project. If solved, it would provide a solution to a huge hurdle in pharmaceutical development.

"We are hoping that we can find some new entry points into the cell to deliver the protein," Silver said.

If successful, such a template could be adapted to develop other proteins.

"The vision is to have a test bed where we can deploy lots of different styles of these proteins and find out which are the best," Silver said. "The ultimate dream would be to design totally new proteins, never seen before. The work can then become a new platform for designing proteins."

Beyond the battlefield

In December 2018, the team was awarded a five-year cooperative agreement worth up to $14.8 million from the Defense Advance Research Projects Agency (DARPA) to pursue the idea.

The initial application of the protein-based compounds would be to halt bleeding and tissue necrosis in traumatic injuries. The use of a compound that halts cell death would allow for transportation and treatment while averting the disseminated tissue damage, infection and cell death that occurs when treatment of such injuries is delayed.

But the long-term, and far more ambitious, goal is to extend the therapeutic benefit of such compounds far beyond the battlefield, Silver said.

Two conditions that stand to benefit from such an approach are heart attacks and strokes.

In the case of heart attacks, for example, even when patients are treated relatively quickly, the myocardial infarction has already killed cells in the oxygen-starved heart muscle. The longer the delay, the more extensive the damage and the greater the radius of tissue death. The "time is muscle" adage refers to the notion that even small delays in treatment can cause irreversible muscle loss, which in turn leads to long-term cardiac damage, the eventual loss of heart muscle function, heart failure and, in extreme cases, death.

The same is true for brain insults, such as strokes or traumatic injuries, where damage to a tiny area of the brain can ripple out to affect surrounding nerve cells. If people in the throes of a heart attack or a stroke could be treated with protein-derived agents that effectively pause ongoing damage until patients are transported to the hospital, their outcomes could be dramatically improved. Such treatment would be particularly useful for injuries occurring in geographically isolated areas where emergency or specialized trauma care is not available.

A biopausing compound could also allow for refrigeration-free preservation of protein-based drugs, enabling easier and cheaper transportation, the team said. Another possible use would be more effective cryopreservation of eggs for in-vitro fertilization. Current oocyte-preservation techniques involve classic deep freezing (cryopreservation) and a newer approach called vitrification, a process that puts tissues in a chemically induced crystallized state. Vitrification requires the use of several chemicals, including ethylene glycol, the active ingredient in antifreeze. While vitrification increases egg viability, data on long-term outcomes for offspring are still lacking.

"A biological that is engineered to avoid toxicity based on a design leveraging millions of years of evolution to confer cellular protection is not only likely to be safer but also to potentially offer improved egg viability over existing technology," Chang said. Similar potential exists for the preservation of organs for transplantation and could complement emerging techniques for whole-body preservation, he added.

Cryptobiotic compounds could also enhance research efforts by allowing scientists to perform targeted silencing of specific proteins or cell types during experiments. For example, if a researcher seeks to determine whether protein A is sensitive to an experimental drug, she could use an IDP-based compound to de-activate the protein in question and then gauge how the cell responds to a given drug in the absence of the silenced protein.

Although it sounds like the stuff of science fiction, the concept makes elegant evolutionary sense. After all, the team's efforts are predicated on adapting preexisting biological recipes tried and tested over tens of millions of years of evolution.

"What we do as synthetic biologists is we look at nature," Silver said. "Then we ask what we can draw from it and use as a platform to solve a problem."

"If somebody told you, let's take a multicellular organism, an animal or a human, and change its metabolism so that it can withstand huge amounts of stress—heat, cold, dehydration—and then bring it back to life, you'd say that's just science fiction, wouldn't you?" Marks said. "But the fact is that real life has already done this."


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Mark Looney, M.D.

Pulmonary and Critical Care Medicine, acute lung injury, acute respiratory distress syndrome, blood transfusions, transfusion-related acute lung injury, neutrophils, neutrophil extracellular traps, platelets, lung transplantation

My laboratory is broadly interesting in study innate immune biology in the normal and injured lung. Using pre-clinical models of acute lung injury, we have focused on neutrophils and platelets, the latter being a bon a fide immune cell with powerful inflammatory potential. One consequence of platelet-neutrophil interactions is the formation of neutrophil extracellular traps (NETs), which we study in both sterile and pathogen-induced lung injury models. We are determining the mechanisms by which platelets trigger NETs and novel pathways to target NETs—which we have discovered are overall barrier disruptive in the lung.

We also use two-photon intravital lung microscopy as a tool for discovery. Using this technique, we have determined that the lung is a major source of mature platelet production in mice. Furthermore, megakaryocytes reside in the extravascular lung and may have potent local immune effects. The lung also contains a wide-range of hematopoietic progenitors, which have the capacity to leave the lung and engraft in the bone marrow for multi-lineage blood production. We are determining the niche-promoting factors responsible for hematopoietic progenitor residence in the lung and the contributions of these cells to the local immune repertoire.

We have an expanding interest in lung transplantation studies, including ischemia-reperfusion injury (primary graft dysfunction) and modeling chronic lung allograft dysfunction (bronchiolitis obliterans). We use the mouse single lung transplantation technique for these studies and to create lung chimeras for investigation.


Antibody “Immunogenicity” may Reflect Homeostasis

It seems counterintuitive that fully human sequence derived antibodies would cause immune responses in humans. Immunoglobulins occur at mg/ml quantities in serum and initiate expression very early in ontogeny, when tolerance to self proteins is imprinted on the nascent immune system. 52 CD4 + T cells are tolerized or deleted in the thymus during development, and are anergized and deleted in the periphery upon contact with inappropriately expressed antigen. 53 , 54 Multiple differential subsets of regulatory T cells control inappropriate responses to antigen in the periphery. 55 , 56 B cells are tolerized by deletional and anergistic mechanisms during development, but can also rescue themselves by a process of receptor editing. 57 Receptor editing is the developmental process where B cells can rearrange their V region segments a second time, thereby altering specificity to avoid deletion. 58 , 59 However, in spite of these mechanisms, human serum from non-diseased normal donors contains detectable levels of anti-idiotype antibody to a wide variety of autoantibodies. 60 – 65 Anti-idiotype antibodies are akin to anti-drug antibodies against therapeutic mAbs, that is, they are antibodies with specificity for the unique V region of other immunoglobulin molecules. The presence of autoantibodies indicates that the tolerance system is not perfect and in fact, poly-reactivity to autoantigens re-emerges during the somatic hypermutation that takes place during an immune response. 66 Anti-idiotype antibodies specific for the newly arising potentially problematic poly-reactive antibodies may represent an additional mechanism for assuring tolerance to self proteins. Indeed, for certain autoimmune diseases, these IgG anti-idiotype antibodies may be protective as the absence of an anti-idiotypic response correlates with the presence of disease in Type I diabetes. 62 The presence of anti-idiotype antibodies is one proposed mechanism for the efficacy of IVIg in so many different autoimmune diseases. 64 , 65 Therefore, mounting an antibody-mediated immune response directed at the combining sites of other antibodies likely is a normal, non-pathogenic event. 67 , 68

If antibody-specific CD4 + T helper cells can be activated in normal donors, even under steady-state conditions that lead to non-pathogenic, anti-autoimmune antibodies, then it should be no surprise that administering large quantities of antibody carrying a single specificity could induce anti-idiotype neutralizing IgG responses in some patients. This effect would be especially pronounced if the antibody happens to carry along a CD4 + helper T cell epitope presentable by the patients’ HLA class II molecules in its V region. These types of immune responses to therapeutic antibodies are of most concern as they tend not to diminish with time and can impact efficacy and associate with pharmacokinetic and safety issues. 69 , 70 It is only when autoantibodies, or anti-therapeutic antibody responses, induce clinical sequelae that we pay attention.


Discussion

We have previously shown that deletion of SARS-CoV E gene leads to an attenuated virus that is a promising vaccine candidate [30–34]. However, since safety and stability are main concerns of live attenuated vaccine candidates, we focused on rSARS-CoV-∆E stability in vitro and in vivo and on the generation of a safe vaccine candidate by identifying the mechanisms of reversion to virulence.

Unexpectedly, serial passage of rSARS-CoV-∆E in cell culture resulted in the generation of chimeric proteins composed of a partial duplication of the membrane gene fused to a part of the leader sequence. Our results are in agreement with a recombinant MHV lacking the E protein (rMHV-∆E) that was viable but its replication was drastically impaired [60]. rMHV-∆E replicated to 10,000-fold lower titers than the parental virus and remarkably, evolved similarly to SARS-CoV-∆E after serial passage in tissue culture cells [61]. Despite that the generation of a chimeric protein was previously observed in MHV when E gene was deleted [61], the presence of a PBM motif within the inserted chimeric sequence and its main role in providing genetic stability to SARS-CoV-ΔE during passage described in this manuscript, were not previously noticed. Alteration of coronavirus genome was not unexpected due to the high frequency of RNA recombination and mutation described for these viruses, both in cell culture and in animals [37, 62–64].

All chimeric SARS-CoV M proteins generated in cell culture were expressed, enhancing virus growth in cell culture compared to rSARS-CoV-∆E. Similarly, rMHV-∆E that contained chimeric proteins also showed significant increases in viral yields [61, 65]. In contrast, mice infected with rSARS-CoVs containing chimeric proteins generated in cell culture showed a decrease in viral titers in the lungs of infected mice even when compared with rSARS-CoV-∆E virus. Similarly, extensive passage in cell culture of other CoVs, including porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), led to less pathogenic strains compared to wild-type viruses, possibly due to the emergence of deletion mutants that lost sequence domain not needed for their growth in cell culture, but that influenced their tropism and in vivo replication [66–68]. Chimeric proteins containing a PBM inserted in the viral genome after passage increased viral fitness in cell culture in a tissue specific manner, i.e., the chimeric protein inserted into viral genome during passage in monkey cells promoted virus growth in cells from this species, whereas the one inserted during passage in murine cells specifically increased virus fitness in cells from mice. Furthermore, these chimeric proteins did not enhance viral growth nor virulence in vivo. These data indicated that the insertion of chimeric proteins specifically adapted the virus for an optimum growth in cell culture but did not enhance in vivo growth nor virulence. In this context, it is also important to note that the activity of PBMs is dependent on their specific sequence, and also on the sequence context in which they are inserted [27, 28, 69, 70].

Serial passage of rSARS-CoV-∆E in mice introduced a partial duplication of 45 nucleotides in the 8a protein, resulting in its reversion to a virulent phenotype. This phenotype was associated to its ability to activate p38 MAPK and to the induction of inflammatory cytokine expression and increased lung damage, as previously described [24, 71].

8a protein is a short transmembrane protein composed of 39 amino acids that forms cation-selective ion channels [72]. SARS-CoV variants with deletions in 8a ORF, have been transmitted and maintained in humans in the late phases of SARS-CoV epidemic [73, 74]. Interestingly, an 8a protein mutant generated during virus passage in vivo contained a new potential PBM (CTTV) localized in the internal region of the carboxy-terminal domain of the protein (Fig 5). Despite the CTVV sequence was already present in the original 8a protein, it most likely does not represent a functional PBM, as it is located within the transmembrane domain of the 8a protein. Active PBMs are in general located in exposed regions of the proteins, usually the end of the carboxy-terminus or, exceptionally, in internal positions within the carboxy-terminal domain, allowing their interaction with PDZ domains, such as it has been observed in the NS5 proteins of tick-borne encephalitis virus (TBEV) and Dengue virus [75, 76]. However, PBMs forming part of a transmembrane domain are not accessible to PDZ-containing proteins and have not been described [29]. Therefore, the new CTVV sequence placed in an exposed environment may constitute a novel and active PBM. The PBM insertion within 8a protein after passaging in vivo could be due to the fact that the ORF8 is one of the regions where most variations were observed between human and animal isolates of SARS-CoV [4]. In fact, a complete genome sequence of SARS-like coronaviruses in bats isolates showed the presence of a PBM within the ORF8 [5]. As mentioned above, species of bats are a natural host of coronaviruses closely related to those responsible for the SARS outbreak.

PDZ domains are among the modules most frequently involved in protein-protein interactions found in all metazoans [69]. In the human genome, there are more than 900 PDZ domains in at least 400 different proteins [77]. Many pathogenic viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, such as hepatitis B virus, influenza virus, rabies virus and human immunodeficiency virus, influencing their replication, dissemination in the host, transmission and virulence [29]. Generation of new proteins containing a PBM after passaging in cell culture and in mice may affect their interaction with a wide range of cellular PDZ-containing proteins, affecting diverse biological functions with high relevance in pathogenesis. E protein PBM participates in two different and independent issues, virus stability and virulence. Our data suggest that when the PBM was present in a proper environment at the end of the E protein, either a native or a mutant protein, viruses remained stable. An independent observation is that the presence of a PBM within E protein confers pathogenicity to the virus [24]. This virulence is prevented either by PBM removal [24] or by the introduction of small deletions within the carboxy-terminus of E protein [53], which by themselves may cause attenuation or, alternatively, by indirectly affecting the PBM. Our results highlighted the critical requirement of viral proteins containing a PBM in the generation of CoVs with virulent phenotypes, and opened up new approaches for the rational design of genetically stable vaccines.

Maintaining the attenuated phenotype of the vaccine candidate after passage in vitro was crucial to avoid the reversion to a virulent phenotype during the design and production of a genetically stable vaccine candidate. To this end, the identification of the relevance of the presence of a functional PBM motif at the carboxy-terminus of a transmembrane protein of the virus has been instrumental in the development of a stable SARS-CoV vaccine candidate. To minimize the risk of regain of virulence after passage, we engineered viruses with small deletions in E gene, instead of deletion of the entire E gene. By preserving the PBM, we observed no evidence for the development of chimeric proteins and thus no gain in virulence. As additional measures to ensure safety of this live attenuated vaccine candidate, we incorporated attenuating mutations into nsp1, in the context of the rSARS-CoV-ΔE or EΔ3. Nsp1 was chosen as a second attenuation target because this gene is located at a distant site (>20 kb) from that of the E gene in the viral genome, making it very unlikely that a single recombination event with a circulating wt coronavirus could result in the restoration of a virulent phenotype.

To analyze the role of SARS-CoV nsp1 in the pathogenesis of the virus, recombinant viruses encoding four different small deletions were generated. Deletion of amino acids 121–129 and 154–165, in the carboxy terminal region of nsp1 led to virus attenuation, indicating that nsp1 enhanced virus pathogenicity, as was previously shown for MHV [45, 48, 49]. Interestingly, these attenuated mutants grew in mice to lower titers than rSARS-CoV, probably by inducing higher IFN responses, indicating that these regions of nsp1 are critical for IFN antagonism. The induction of a higher innate immune response by the nsp1 deletion is most probably responsible for the decrease in SARS-CoV-nsp1* virus titers observed in mice and, to a lesser extent, in DBT-mACE–2 cells. In fact, a rSARS-CoV lacking the nsp1 protein grew poorly in IFN competent cells, but replicated as efficiently as the wt virus in IFN deficient cells [46], consistent with our findings. Similarly, titers of MHV deleted in nsp1 are restored almost to wild type levels in type I IFN receptor-deficient mice [48].

Immunization with singly deleted rSARS-CoV protected mice against challenge with rSARS-CoV, as it was previously shown with MHV nsp1-deletion mutants [48, 49]. SARS-CoV-nsp1ΔD-EΔ3, which contained deletions in nsp1 and E protein, maintained its attenuated phenotype after passage in Vero E6 cells and in mice. In addition, immunization with this double mutant fully protected mice from challenge with the parental virulent virus, indicating that it is a promising vaccine candidate in terms of both stability and efficacy.

Both humoral and cellular responses are relevant to protect from SARS [18, 19, 78, 79]. The viruses generated in this work express all viral proteins, except for small regions deleted in the E and nsp1 proteins, therefore have the potential of inducing both antibody and T cell responses, making this type of live vaccine more attractive than subunit or non replicating virus vaccines. Understanding of the molecular mechanisms by which an attenuated SARS-CoV reverted to a virulent phenotype could also be applied to the development of other relevant CoVs vaccines, such as MERS-CoV.


The Feasibility of Genetic Engineering to Produce More Potent Lantibiotics

RiPPs Recombinant Expression for Classical Engineering—Nisin as an Example

A wide range of technologies are at our disposal for producing a peptide such as chemical synthesis, recombinant DNA technologies, and in vitro translation systems. The choice of a strategy for gene-encoded peptide production is largely determined by their size and chemical complexity. Peptides with sophisticated PTMs-generated structures such as RiPPs are preferably produced biosynthetically using host expression cells. Host cells with installed and functional PTM enzymes should be able to express RiPPs in an intact active form with distinctive architecture dominated by lanthionine bridges. In this context, the absence of a widely used large-scale production platform for lantibiotics with consistent quality is one of the main drawbacks of their therapeutic application.

There are generally two options for recombinant expression of lantibiotics, homologous or heterologous expression systems. Homologous expression strategies have limited use in particular due to the difficulty of cultivating the original producers (Maffioli et al., 2015 Mohr et al., 2015) and/or the difficulty of lantibiotics expression induction under laboratory conditions (Wescombe et al., 2011 Garg et al., 2012). Conversely, heterologous biosynthesis with host cells such as Lactococcus lactis, Bacillus subtilis, Bacillus cereus, and E. coli are normally preferred and, in many cases, well established. So far, several genetically manipulatable Streptomyces strains, including Streptomyces lividans (Ahmed et al., 2020), S. albus (Myronovskyi et al., 2018), S. coelicolor M145 (Gomez-Escribano and Bibb, 2011), S. venezuelae ATCC 15439 (Kim et al., 2015), S. avermitilis (Komatsu et al., 2010) have been reported to synthesize natural products from genetically engineered biosynthetic gene clusters. However, Escherichia coli is still the most common and attractive option. A list of successful examples of recombinant expression of different RiPPs families in heterologous hosts such as E. coli and Streptomyces strains were recently compiled by Zhang and colleagues (Zhang et al., 2018). Heterologous production of lantibiotics is generally conducted as inactive form of the antimicrobial peptide (pre-lantibiotic) to prevent any detrimental effects on host cell growth and viability.

In addition, there is great interest to express lantibiotics in microbial host that should facilitate a straightforward and efficient engineering (Kuipers et al., 1996). Numerous conventional protein engineering approaches such as site-directed mutagenesis, directed evolution and various computational tools aid synthetic biologists and biochemists to selectively diversify the properties of expressed therapeutic peptides. These include manipulations of thermodynamic stability, increased bioavailability, reduced aggregation or enhanced specificity and proteolytic stability (Adhikari et al., 2019). In addition, the implementation of in vitro and in vivo approaches in combination with genome mining data and high-throughput screening strategies has opened up unprecedented opportunities to modify and even improve antimicrobial activity, manipulate the physicochemical properties and widening of the antibacterial spectrum in the production of lantibiotics (Field et al., 2015). Recently an attempt to design and biosynthesize a two-lipid II binding motifs-containing lantibiotic, called TL19, is the latest example showing 64-fold stronger activity against Enterococcus faecium than nisin (Zhao et al., 2020).

The potential of classical protein engineering in complex RiPPs is perhaps best illustrated by recombinant nisin, well known for its broad-spectrum antibacterial activity produced in certain strains of L. lactis (Lubelski et al., 2008). Nisin as the most widely used lantibiotic in the food industry over the past 50 years has undergone various methods of bioengineering with an aim to improving its function and/or physicochemical features (Shin et al., 2016). To expand the scope of its activity, several bioengineered variants of nisin have been generated by site-directed mutagenesis and by classical chemical modifications (Ross et al., 2012), including in vitro chemical synthesis (Knerr and van der Donk, 2012 Koopmans et al., 2015). For example, the use of the saturation mutagenesis approach has led to the production of nisin S29 derivatives with increased activity against a number of Gram-positive antibiotic-resistant pathogens. In addition, the increased antimicrobial activity was generated in comparison to the wild type nisin A when tested against Gram-negative food-borne pathogens (Field et al., 2012). Moreover, recombinant production of the nisin Z mutants N20K, M21K, N27K, H31K improved peptide solubility at alkaline pH (Rollema et al., 1995). Next, residue alterations at distinct locations enabled the improvement of antimicrobial activity (Islam et al., 2009 Healy et al., 2013 Geng and Smith, 2018), the enhancement of diffusion through complex polymers (Rouse et al., 2012), and widening effect on some Gram-negative bacteria (Field et al., 2012).

Studies on the effects of the hinge region (NMK) length between rings C and D on antimicrobial activity and host specificity of nisin was also performed (Zhou et al., 2015). Although most variants with shorter or larger hinge length are less active than the wild type, some variants (+2, +1, 𢄡, 𢄢) exhibited higher antimicrobial activity than the wild type nisin A in agar-well-diffusion assays against L. lactis MG1363, Listeria monocytogenes, Enterococcus faecalis VE14089, Bacillus sporothermodurans IC4, and Bacillus cereus 4153. In addition, an extended nisin A variant of the hinge region (20NMKIV24) has been introduced, bypassing the human pathogen’s lantibiotic resistance while showing a slight decrease in antimicrobial activity (Zaschke-Kriesche et al., 2019). In this context, Figure 3 represents different variants of nisin produced by classical protein engineering together with a graphical representation of the mechanism of action of this bioactive peptide.

Figure 3. Nisin is one of the best-studied ribosomally synthesized, pore-forming, cationic, antimicrobial peptides. (A) Sequence comparison of three nisin variants from L. lactis and marking the important regions of nisin targeted for bioengineering by classical molecular biology approaches (Chatterjee et al., 2005) (B) two-steps mode of action of nisin.

However, the expression of lantibiotics in popular prokaryotic hosts like E. coli is challenging as these bacteria have no enzymes to perform suitable PTMs to convert pro-peptide into a mature bioactive peptide. Therefore, their recombinant expression has to be coupled with the co-expression of active PTM biosynthetic gene clusters either in vitro or in vivo. These strategies are not in the focus of our study and interested readers are directed to numerous studies and reviews dedicated to this topic (Zhang et al., 2018 Myronovskyi and Luzhetskyy, 2019). We are mainly concerned with the in vivo design strategies that focus to expand the functional scope of AMPs from ribosomal templates beyond the classical protein engineering approaches.

Beyond Classical Protein Engineering: Expanding the Scope of Protein Translation

It was argued that chemical and functional diversity delivered by PTMs in AMPs such as lantibiotics can be further supplemented or even expanded by the co-translational insertion of non-canonical amino acids (ncAAs) (Budisa, 2013). Indeed, a limited and conservative set of 20 canonical amino acids used by ribosomes to encode polypeptides in nature usually does not cover enough chemical space required to substantially expand their functional and structural diversity. In nature, this is normally achieved by site-selective PTMs that create special non-canonical amino acid side chains such as dehydroalanine (Dha) and dehydrobutyrine (Dhb) in lantibiotics (Figure 3). To directly mimic these and similar PTMs, chemists used, e.g., rhodium-catalyzed arylations (Key and Miller, 2017), P450-catalyzed cyclopropanations (Gober et al., 2017), and photocatalytic activation (de Bruijn and Roelfes, 2018) in thiopeptides, as well as metalloporphyrin-catalyzed alkylation of methionine in nisin (Maaskant and Roelfes, 2019) as attempts to obtaining novel AMPs with improved properties and/or activities. However, it is difficult to mimic PTM machinery chemically, whereas classical peptide synthetic protocols such as solid-phase peptide synthesis (SPPS) could not cover the chemical complexity of these natural products.

Therefore, the use of recombinant DNA technology that operates with heterologous expression of biosynthetic gene clusters and enriched with reprogrammed protein translation (Figure 4) can provide a reasonable solution to the above-mentioned problems (Hoesl and Budisa, 2012). It enables specific insertion of the biological, chemical, and physical properties delivered by ncAAs that can be accurately defined by the chemist at the bench. Cells equipped with various bioorthogonal chemistries have the potential to perform catalytic transformations that are not found in biology, including the creation of new metabolic and informational pathways (Devaraj, 2018). These modifications can also serve as redox sensors, spectroscopic markers (e.g., spin-labeling) or sensors of protein-ligand interactions. Metal-binding amino acids could lead to new structural, catalytic, or regulatory elements in proteins, while diazirines allow site-specific photo-crosslinking of the target protein to its substrate. Photoactive and photo-isomerizable ncAAs with novel photo-physical properties such as azobenzene side chains can be used to photo-regulate protein activity. Similarly, photocaged ncAAs can be activated by light to turn on enzymatic activity spatiotemporally (for review see Young and Schultz, 2010).

Figure 4. The radial arrangement of the standard genetic code in RNA format (A) with the side chains of 20 canonical amino acids (cAA). The standard amino acid repertoire of the genetic code can be modified or expanded (B) by non-canonical amino acids (ncAAs). By reprogramming protein translation, the ncAAs can be incorporated into recombinant proteins using in vivo and in vitro methods. These insertions offer useful modifications that can be made to the protein main chain (backbone) and the amino acid side chains (aliphatic or aromatic). Modified backbones and side chains often have unique physicochemical properties with the potential to dramatically expand the chemical and functional space of ribosomally synthesized peptides and proteins.

Directed evolution of aminoacyl-tRNA synthase (aaRS) plays a key role in the generation of such molecular tools. Desired ncAAs can be incorporated in a site-directed manner by reading in-frame stop codons, usually amber stop codons (UAG) with a suitable orthogonal pair (o-pair). Such a pair consists of an aaRS enzyme to activate the ncAA and its associated orthogonal tRNA (vide infra). However, the transition to recombinant production with such a possibility by using heterologous expression hosts is not trivial. For that reason, the residue-specific replacement of a particular amino acid at all positions in a protein sequence (known as selective pressure incorporation, vide infra) is a reasonable alternative as it does not require o-pairs (Budisa, 2004). Finally, the capacity of ncAAs incorporation should not interfere with the sequence of posttranslational modifications leading to the formation of lantibiotics and the restoration of their bioactivity.

With such systems in hands we would have very sophisticated tools to rationally engineer RiPPs scaffolds as now they can be redesigned by reprogrammed protein translation which utilize various ncAAs. This approach would have many advantages over the above discussed classical chemical or recombinant approaches as it enables: (i) genetic encoding of desired ncAAs and its sequence positioning (ii) recombinant production under mild conditions at room temperature and atmospheric pressure (iii) the targeted functionalization (e.g., various site-directed bioconjugations, light induced cross-linking, metal or cofactor binding, fluorophore or pharmacophore attachment, adhesiveness, etc.) (Cropp and Schultz, 2004). Crucial requirements to expand the scope of protein synthesis with ncAAs should be cellular uptake, their intracellular metabolic stability and translational activity (i.e., incorporation). Finally, it is necessary to reallocate (reassign) coding triplets (codons) in the genetic code to insert ncAAs in target protein/peptide sequences.


References and Notes

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The discovery of BMAA, and examples of biomagnifications and protein incorporation involving other non-protein amino acids. Amyotroph Lateral Scler 10(suppl 2):21-25 2009 . http://www.ncbi.nlm.nih.gov/pubmed/19929727

Distribution and toxicity of α-amino-β-methylaminopropionic acid. Fed Proc 31(5):1473-1475 1972 . http://www.ncbi.nlm.nih.gov/pubmed/5056173

Selective degeneration of cerebellar cortical neurons caused by cycad neurotoxin, ʟ-β-methylaminoalanine (ʟ-BMAA), in rats. Neuropathol Appl Neurobiol 16(2):153-169 1990 . http://dx.doi.org/10.1111/j.1365-2990.1990.tb00944.x

2-Amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour: an unlikely cause of amyotrophic lateral sclerosis and parkinsonism-dementia of Guam. Neurology 40(5):767-772 1990 . http://www.ncbi.nlm.nih.gov/pubmed/2330104

Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam. Neurology 58(6):956-959 2002 . http://www.ncbi.nlm.nih.gov/pubmed/11914415

Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci USA 100(23):13380-13383 2003 . http://dx.doi.org/10.1073/pnas.2235808100

Biomagnification of cycad neurotoxins in flying foxes: implications for ALS-PDC in Guam. Neurology 61(3):387-389 2003 . http://dx.doi.org/10.1212/01.WNL.0000078320.18564.9F

Occurrence of β-methylamino-ʟ-alanine (BMAA) in ALS/PDC patients from Guam. Acta Neurol Scand 110(4):267-269 2004 . http://dx.doi.org/10.1111/j.1600-0404.2004.00320.x

Blooms like it hot. Science 320(5872):57-58 2008 . http://dx.doi.org/10.1126/science.1155398

Diverse taxa of cyanobacteria produce β-N-methylamino-ʟ-alanine, a neurotoxic amino acid. Proc Natl Acad Sci USA 102(14):5074-5078 2005 . http://dx.doi.org/10.1073/pnas.0501526102

Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease. Acta Neurol Scand 120(4):216-225 2009 . http://dx.doi.org/10.1111/j.1600-0404.2008.01150.x

Sports and trauma in amyotrophic lateral sclerosis revisited. J Neurol Sci 262(1-2):45-53 2007 . http://dx.doi.org/10.1016/j.jns.2007.06.021

An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14(6):1105-1116 1995 . http://dx.doi.org/10.1016/0896-6273(95)90259-7

Lack of cerebral BMAA in human cerebral cortex. Neurology 72 (15))1360-1361( 2009 . http://dx.doi.org/10.1212/WNL.0b013e3181a0fed1

Lack of β-methylamino-ʟ-alanine in brain from controls, AD, or Chamorros with PDC. Neurology 65(5):768-769 2005 . http://dx.doi.org/10.1212/01.wnl.0000174523.62022.52

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Distinguishing the cyanobacterial neurotoxin β-N-methylamino-ʟ-alanine (BMAA) from other diamino acids. Toxicon 57(5):730-738 2011 . http://dx.doi.org/10.1016/j.toxicon.2011.02.005

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BMAA selectively injures motor neurons via AMPA/kainate receptor activation. Exp Neurol 201(1):244-252 2006 . http://dx.doi.org/10.1016/j.expneurol.2006.04.017

Depletion of reduced glutathione enhances motor neuron degeneration in vitro and in vivo. Neuroscience 144(3):991-1003 2007 . http://dx.doi.org/10.1016/j.neuroscience.2006.09.064

Cyanobacterial blooms and the occurrence of the neurotoxin, beta-N-methylamino-ʟ-alanine (BMAA), in South Florida aquatic food webs. Harmful Algae 9(6):620-635 2010 . http://dx.doi.org/10.1016/j.hal.2010.05.002

Toxin mixture in cyanobacterial blooms—a critical comparison of reality with current procedures employed in human health risk assessment. Adv Exp Med Biol 619:885-912 2008 . http://www.ncbi.nlm.nih.gov/pubmed/18461795

Cyanobacteria and BMAA exposure from desert dust: a possible link to sporadic ALS among Gulf War veterans. Amyotroph Lateral Scler 10(Suppl 2):109-117 2009 . http://dx.doi.org/10.3109/17482960903286066

β-N-methylamino-ʟ-alanine (BMAA) in novel South African cyanobacterial isolates. Ecotoxicol Environ Saf 71(2):309-313 2008 . http://dx.doi.org/10.1016/j.ecoenv.2008.04.010

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Early hippocampal cell death, and late learning and memory deficits in rats exposed to the environmental toxin BMAA (β-N-methylamino-ʟ-alanine) during the neonatal period. Behav Brain Res 219(2):310-20 2011 .


First Life with "Alien" DNA Created in Lab

For billions of years, the history of life has been written with just four letters &mdash A, T, C and G, the labels given to the DNA subunits contained in all organisms. That alphabet has just grown longer, researchers announce, with the creation of a living cell that has two 'foreign' DNA building blocks in its genome.

Hailed as a breakthrough by other scientists, the work is a step towards the synthesis of cells able to churn out drugs and other useful molecules. It also raises the possibility that cells could one day be engineered without any of the four DNA bases used by all organisms on Earth.

&ldquoWhat we have now is a living cell that literally stores increased genetic information,&rdquo says Floyd Romesberg, a chemical biologist at the Scripps Research Institute in La Jolla, California, who led the 15-year effort. Their research appears online today in Nature.

Each strand of the DNA's double helix has a backbone of sugar molecules and, attached to it, chemical subunits known as bases. There are four different bases: adenine (A), thymine (T), cytosine (C) and guanine (G). These letters represent the code for the amino-acid building blocks that make up proteins. The bases bind the two DNA strands together, with an A always bonding to a T on the opposite strand (and vice versa), and C and G doing likewise.

Test-tube letters
Scientists first questioned whether life could store information using other chemical groups in the 1960s. But it wasn&rsquot until 1989 that Steven Benner, then at the Swiss Federal Institute of Technology in Zurich, and his team coaxed modified forms of cytosine and guanine into DNA molecules. In test-tube reactions, strands made of these &ldquofunny letters&rdquo, as Benner calls them, copied themselves and encoded RNA and proteins.

The bases engineered by Romesberg&rsquos team are more alien, bearing little chemical resemblance to the four natural ones, Benner says. In a 2008 paper, and in follow-up experiments, the group reported efforts to pair chemicals together from a list of 60 candidates and screen the 3,600 resulting combinations. They identified a pair of bases, known as d5SICS and dNaM, that looked promising. In particular, the molecules had to be compatible with the enzymatic machinery that copies and translates DNA.

&ldquoWe didn&rsquot even think back then that we could move into an organism with this base pair,&rdquo says Denis Malyshev, a former graduate student in Romesberg&rsquos lab who is first author of the new paper. Working with test-tube reactions, the scientists succeeded in getting their unnatural base pair to copy itself and be transcribed into RNA, which required the bases to be recognized by enzymes that had evolved to use A, T, C and G.

The first challenge to creating this alien life was to get cells to accept the foreign bases needed to maintain the molecule in DNA through repeated rounds of cell division, during which DNA is copied. The team engineered the bacterium Escherichia coli to express a gene from a diatom &mdash a single-celled alga &mdash encoding a protein that allowed the molecules to pass through the bacterium's membrane.

The scientists then created a short loop of DNA, called a plasmid, containing a single pair of the foreign bases, and inserted the whole thing into E. coli cells. With the diatom protein supplying a diet of foreign nucleotides, the plasmid was copied and passed on to dividing E. coli cells for nearly a week. When the supply of foreign nucleotides ran out, the bacteria replaced the foreign bases with natural ones.

Alien control
Malyshev sees the ability to control the uptake of foreign DNA bases as a safety measure that would prevent the survival of alien cells outside the lab, should they escape. But other researchers, including Benner, are trying to engineer cells that can make foreign bases from scratch, obviating the need for a feedstock.

Romesberg&rsquos group is working on getting foreign DNA to encode proteins that contain amino acids other than the 20 that together make up nearly all natural proteins. Amino acids are encoded by 'codons' of three DNA letters apiece, so the addition of just two foreign DNA 'letters' would vastly expand a cell&rsquos ability to encode new amino acids. &ldquoIf you read a book that was written with four letters, you&rsquore not going to be able to tell many interesting stories,&rdquo Romesberg says. &ldquoIf you&rsquore given more letters, you can invent new words, you can find new ways to use those words and you can probably tell more interesting stories.&rdquo

Potential uses of the technology include the incorporation of a toxic amino acid into a protein to ensure that it kills only cancer cells, and the development of glowing amino acids that could help scientists to track biological reactions under the microscope. Romesberg&rsquos team has founded a company called Synthorx in San Diego, California, to commercialize the work.

Ross Thyer, a synthetic biologist at the University of Texas at Austin who co-authored a related News and Views article, says that the work is &ldquoa big leap forward in what we can do&rdquo. It should be possible to get the foreign DNA to encode new amino acids, he says.

&ldquoMany in the broader community thought that Floyd's result would be impossible,&rdquo says Benner, because chemical reactions involving DNA, such as replication, need to be exquisitely sensitive to avoid mutation.

The alien E. coli contains just a single pair of foreign DNA bases out of millions. But Benner sees no reason why a fully alien cell isn&rsquot possible. &ldquoI don&rsquot think there&rsquos any limit,&rdquo he says. &ldquoIf you go back and rerun evolution for four billion years, you could come up with a different genetic system.&rdquo

But creating a wholly synthetic organism would be a huge challenge. &ldquoA lot of times people will say you&rsquoll make an organism completely out of your unnatural DNA,&rdquo says Romesberg. &ldquoThat&rsquos just not going to happen, because there are too many things that recognize DNA. It&rsquos too integrated into every facet of a cell&rsquos life.&rdquo

This article is reproduced with permission from the magazine Nature. The article was first published on May 7, 2014.


Conclusion

It has been a great pleasure to see our science grow in many new and exciting ways over the past few years. I have been fortunate to have very talented people come to my laboratory from a wide range of backgrounds, from total chemical synthesis to transgenic animals, through biochemistry, genetics, molecular evolution, structural biology and cell biology, and to be surrounded by some great collaborators. It has been a great pleasure to learn from everyone in the laboratory and to watch people in the laboratory learn from each other.

I believe that we are training a new generation of scientists who can seamlessly engineer across a range of scales from molecules to systems. They can engineer the specificity of biological networks and biological molecules as well as control the structures of small molecules atom by atom. By coupling these abilities, we have begun to provide solutions to problems previously viewed as intractable. I hope that the people that invested in training me get satisfaction from seeing the fragments of everything I learned from them fused to create something new, and I look forward to being surprised and excited by what the extraordinarily talented alumni that are beginning to emerge from my laboratory may do in the future.


Scientists direct bacteria with expanded genetic code to evolve extreme heat tolerance

Escherichia coli. Credit: Rocky Mountain Laboratories, NIAID, NIH

In recent years, scientists have engineered bacteria with expanded genetic codes that produce proteins made from a wider range of molecular building blocks, opening up a promising front in protein engineering.

Now, Scripps Research scientists have shown that such synthetic bacteria can evolve proteins in the laboratory with enhanced properties using mechanisms that might not be possible with nature's 20 amino acid building blocks.

Exposing bacteria with an artificially expanded genetic code to temperatures at which they cannot normally grow, the researchers found that some of the bacteria evolved new heat-resistant proteins that remain stable at temperatures where they would typically inactivate. The researchers reported their findings in the Journal of the American Chemical Society (JACS).

Virtually every organism on earth uses the same 20 amino acids as the building blocks to make proteins—the large molecules that carry out the majority of cellular functions. Peter Schultz, Ph.D., the senior author of the JACS paper and president and CEO of Scripps Research, pioneered a method to reprogram the cell's own protein biosynthetic machinery to add new amino acids to proteins, termed non-canonical amino acids (ncAAs), with chemical structures and properties not found in the common 20 amino acids.

This expanded genetic code has been used in the past to rationally design proteins with novel properties for use as tools to study how proteins work in cells and as new precision-engineered drugs for cancer. The researchers now asked whether synthetic bacteria with expanded genetic codes have an evolutionary advantage over those that are limited to 20 building blocks—is a 21 amino acid code better than a 20 amino acid code from an evolutionary fitness perspective?

"Ever since we first expanded the range of amino acids that can be incorporated in proteins, much work has gone into using these systems to engineer molecules with new or enhanced properties," says Schultz. "Here, we've shown that combining an expanded genetic code with a laboratory evolution one can create proteins with enhanced properties that may not be readily achievable with nature's more limited set."

The scientists started by tweaking the genome of E. coli so that the bacteria could produce the protein homoserine o-succinyltransferase (metA) using a 21 amino acid code instead of the common 20 amino acid code. An important metabolic enzyme, metA dictates the maximum temperature at which E. coli can thrive. Above that temperature, metA begins to inactivate and the bacteria die. The researchers then made mutants of metA, in which almost any amino acid in the natural protein could be replaced with a 21st noncanonical amino acid.

At this point, they let natural selection—the central mechanism of evolution—work its magic. By heating the bacteria to 44 degrees Celsius—a temperature at which normal metA protein cannot function, and as a consequence, bacteria cannot grow—the scientists put selective pressure on the bacteria population. As expected, some of the mutant bacteria were able to survive beyond their typical temperature ceiling, thanks to possessing a mutant metA that was more heat stable—all other bacteria died.

In this way, the researchers were able to drive the bacteria to evolve a mutant metA enzyme that could withstand temperatures 21 degrees higher than normal, nearly twice the thermal stability increase that people typically achieve when restricted to mutations limited to the common 20 amino acid building blocks.

The researchers then identified the specific genetic sequence change that resulted in the mutant metA and found it was due to the unique chemical properties of one of their noncanonical amino acids that laboratory evolution exploited in a clever way to stabilize the protein.

"It's striking how making such a small mutation with a new amino acid not present in nature leads to such a significant improvement in the physical properties of the protein," says Schultz.

"This experiment raises the question of whether a 20 amino acid code is the optimal genetic code—if we discover life forms with expanded codes will they have an evolutionary advantage, and what would we be like if God had worked on the seventh day and added a few more amino acids to the code?"


Watch the video: Αμινοξέα - BCAA - Ό,τι χρειάζεται να ξέρετε! (November 2022).