Biogenesis and engineering of interleukin 12 family cytokines.

Interleukin 12 (IL-12) family cytokines are secreted proteins that regulate immune responses. Each family member is a heterodimer and nature uses shared building blocks to assemble the functionally distinct IL-12 cytokines. In recent years we have gained insights into the molecular principles and cellular regulation of IL-12 family biogenesis. For each of the family members, generally one subunit depends on its partner to acquire its native structure and be secreted from immune cells. If unpaired, molecular chaperones retain these subunits in cells. This allows cells to regulate and control secretion of the highly potent IL-12 family cytokines. Molecular insights gained into IL-12 family biogenesis, structure, and function now allow us to engineer IL-12 family cytokines to develop novel immunotherapeutic approaches.

Development of an enabling platform biotechnology for the production of proteins.

Protein-based drugs are a mainstay of modern medicine. In contrast to antibodies, most of these need highly individualized production processes which often limits their development. Here, we develop an immunoglobulin domain tag (i-Tag), which can be fused to any protein of interest. This tag is made of a linear arrangement of antibody light chain constant domains. It enhances expression as well as secretion of the fusion partner and allows for simple purification of several structurally and functionally distinct fusion proteins. Furthermore, it improves the biophysical characteristics of most fusion proteins tested, is inert, and does not compromise the fusion partners' functionality. Taken together, the i-Tag should facilitate the development of biopharmaceuticals and diagnostic proteins otherwise lacking a common structural element.

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines.

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and ß subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate ß subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone-client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:ß assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.

A folding switch regulates interleukin 27 biogenesis and secretion of its α-subunit as a cytokine.

A common design principle of heteromeric signaling proteins is the use of shared subunits. This allows encoding of complex messages while maintaining evolutionary flexibility. How cells regulate and control assembly of such composite signaling proteins remains an important open question. An example of particular complexity and biological relevance is the interleukin 12 (IL-12) family. Four functionally distinct αß heterodimers are assembled from only five subunits to regulate immune cell function and development. In addition, some subunits act as independent signaling molecules. Here we unveil key molecular mechanisms governing IL-27 biogenesis, an IL-12 family member that limits infections and autoimmunity. In mice, the IL-27α subunit is secreted as a cytokine, whereas in humans only heterodimeric IL-27 is present. Surprisingly, we find that differences in a single amino acid determine if IL-27α can be secreted autonomously, acting as a signaling molecule, or if it depends on heterodimerization for secretion. By combining computer simulations with biochemical experiments, we dissect the underlying structural determinants: a protein folding switch coupled to disulfide bond formation regulates chaperone-mediated retention versus secretion. Using these insights, we rationally change folding and assembly control for this protein. This provides the basis for a more human-like IL-27 system in mice and establishes a secretion-competent human IL-27α that signals on its own and can regulate immune cell function. Taken together, our data reveal a close link between protein folding and immunoregulation. Insights into the underlying mechanisms can be used to engineer immune modulators.

Assembly-induced folding regulates interleukin 12 biogenesis and secretion.

Members of the IL-12 family perform essential functions in immunoregulation by connecting innate and adaptive immunity and are emerging therapeutic targets. They are unique among other interleukins in forming heterodimers that arise from extensive subunit sharing within the family, leading to the production of at least four functionally distinct heterodimers from only five subunits. This raises important questions about how the assembly of IL-12 family members is regulated and controlled in the cell. Here, using cell-biological approaches, we have dissected basic principles that underlie the biogenesis of the founding member of the family, IL-12. Within the native IL-12 heterodimer, composed of IL-12α and IL-12ß, IL-12α possesses three intramolecular and one intermolecular disulfide bridges. We show that, in isolation, IL-12α fails to form its native structure but, instead, misfolds, forming incorrect disulfide bonds. Co-expression of its ß subunit inhibits misfolding and thus allows secretion of biologically active heterodimeric IL-12. On the basis of these findings, we identified the disulfide bonds in IL-12α that are critical for assembly-induced secretion and biological activity of IL-12 versus misfolding and degradation of IL-12α. Surprisingly, two of the three disulfide bridges in IL-12α are dispensable for IL-12 secretion, stability, and biological activity. Extending our findings, we show that misfolding also occurs for IL-23α, another IL-12 family protein. Our results indicate that assembly-induced folding is key in IL-12 family biogenesis and secretion. The identification of essential disulfide bonds that underlie this process lays the basis for a simplified yet functional IL-12 cytokine.

Modeling of the human interleukin 12:receptor complex allows to engineer attenuated cytokine variants.

Interleukin 12 (IL-12) plays major roles in immune defense against intracellular pathogens. By activating T cells and increasing antigen presentation, it is also a very potent anti-tumor molecule. Strong immune activation and systemic toxicity, however, so far limit its potential therapeutic use. Building on recent experimental structures of IL-12 related cytokine:receptor complexes, we here provide a high-resolution computational model of the human IL-12:receptor complex. We design attenuated IL-12 variants with lower receptor binding affinities based on molecular dynamics simulations, and subsequently validate them experimentally. These variants show reduced activation of natural killer cells while maintaining T cell activation. This immunological signature is important to develop IL-12 for cancer treatment, where natural killer cells contribute to severe side-effects. Taken together, our study provides detailed insights into structure and dynamics of the human IL-12:receptor complex and leverages them for engineering attenuated variants to elicit fewer side-effects while maintaining relevant biological activity.

Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

Interleukin 12 (IL-12) family cytokines connect the innate and adaptive branches of the immune system and regulate immune responses. A unique characteristic of this family is that each member is anα:ßheterodimer. For human αsubunits it has been shown that they depend on theirßsubunit for structure formation and secretion from cells. Since subunits are shared within the family and IL-12 as well as IL-23 use the same ßsubunit, subunit competition may influence cytokine secretion and thus downstream immunological functions. Here, we rationally design a folding-competent human IL-23α subunit that does not depend on itsßsubunit for structure formation. This engineered variant still forms a functional heterodimeric cytokine but shows less chaperone dependency and stronger affinity in assembly with its ßsubunit. It forms IL-23 more efficiently than its natural counterpart, skewing the balance of IL-12 and IL-23 towards more IL-23 formation. Together, our study shows that folding-competent human IL-12 familyαsubunits are obtainable by only few mutations and compatible with assembly and function of the cytokine. These findings might suggest that human α subunits have evolved for assembly-dependent folding to maintain and regulate correct IL-12 family member ratios in the light of subunit competition.

Influence of glycosylation on IL-12 family cytokine biogenesis and function.

The interleukin 12 (IL-12) family of cytokines regulates T cell functions and is key for the orchestration of immune responses. Each heterodimeric IL-12 family member is a glycoprotein. However, the impact of glycosylation on biogenesis and function of the different family members has remained incompletely defined. Here, we identify glycosylation sites within human IL-12 family subunits that become modified upon secretion. Building on these insights, we show that glycosylation is dispensable for secretion of human IL-12 family cytokines except for IL-35. Furthermore, our data show that glycosylation differentially influences IL-12 family cytokine functionality, with IL-27 being most strongly affected. Taken together, our study provides a comprehensive analysis of how glycosylation affects biogenesis and function of a key human cytokine family and provides the basis for selectively modulating their secretion via targeting glycosylation.

An Interspecies Analysis Reveals Molecular Construction Principles of Interleukin 27.

Interleukin 27 (IL-27) is a cytokine that regulates inflammatory responses. It is composed of an α subunit (IL-27α) and a ß subunit (EBI3), which together form heterodimeric IL-27. Despite this general principle, IL-27 from different species shows distinct characteristics: Human IL-27α is not secreted autonomously while EBI3 is. In mice, the subunits show a reciprocal behavior. The molecular basis and the evolutionary conservation of these differences have remained unclear. They are biologically important, however, since secreted IL-27 subunits can act as cytokines on their own. Here, we show that formation of a single disulfide bond is an evolutionary conserved trait, which determines secretion-competency of IL-27α. Furthermore, combining cell-biological with computational approaches, we provide detailed structural insights into IL-27 heterodimerization and find that it relies on a conserved interface. Lastly, our study reveals a hitherto unknown construction principle of IL-27: one secretion-competent subunit generally pairs with one that depends on the other to induce its secretion. Taken together, these findings significantly extend our understanding of IL-27 biogenesis as a key cytokine and highlight how protein assembly can influence immunoregulation.

Harvest of the Oleaginous Microalgae Scenedesmus obtusiusculus by Flocculation From Culture Based on Natural Water Sources.

Harvest and dewatering poses a significant economical burden for industrial algae biomass production. To mitigate these effects, energy efficient techniques for these process steps have to be developed. Flocculation of the microalgae Scenedesmus obtusiusculus in salt based medium was induced by pH-shift and alternatively by addition of two biological flocculants, chitosan, and the commercial tannin CFL-PT. This is the first time that CFL-PT is used as an algae flocculant particularly focusing on harvesting of halophilic strains. The method was characterized and subsequently optimized. In comparison to biological flocculants, induction by pH shift is far cheaper, but due to buffering effects of the brackish cultivation medium infeasible amounts of base are required to raise the pH-value. tannin appears to be superior compared to chitosan not only in the absence of algae organic matter (AOM), but tannin-based harvest is also more robust regarding culture pH in presence of AOM. A higher flocculant-demand for modified tannin compared to chitosan is offset by the lower price. Given the employed strain and cultivation conditions, cultivation time had no pronounced effect on flocculation efficiencies (FE) while algae zeta-potential and bacterial communities also remained stable.