A spider silk-derived solubility domain inhibits nuclear and cytosolic protein aggregation in human cells

Due to the inherent toxicity of protein aggregates, the propensity of natural, functional amyloidogenic proteins to aggregate must be tightly controlled to avoid negative consequences on cellular viability. The importance of controlled aggregation in biological processes is illustrated by spidroins, which are functional amyloidogenic proteins that form the basis for spider silk. Premature aggregation of spidroins is prevented by the N-terminal NT domain. Here we explored the potential of the engineered, spidroin-based NT* domain in preventing protein aggregation in the intracellular environment of human cells. We show that the NT* domain increases the soluble pool of a reporter protein carrying a ligand-regulatable aggregation domain. Interestingly, the NT* domain prevents the formation of aggregates independent of its position in the aggregation-prone protein. The ability of the NT* domain to inhibit ligand-regulated aggregation was evident both in the cytosolic and nuclear compartments, which are both highly relevant for human disorders linked to non-physiological protein aggregation. We conclude that the spidroin-derived NT* domain has a generic anti-aggregation activity, independent of position or subcellular location, that is also active in human cells and propose that the NT* domain can potentially be exploited in controlling protein aggregation of disease-associated proteins.

1. More background or discussion on using such a strategy to prevent protein aggregation, particularly disease-related protein aggregation, is needed. In other word, the importance of this study has to be adequately highlighted. 2. What is the benefit of using such a strategy over small molecules, especially from the clinical translation point of view. 3. In addition, the study used a reporter protein whose aggregation is ligand-regulatable. Why not using directly disease-related proteins. 4. The mechanism for the independence of the position of the NT* domain in the host protein is unclear. This should be studied. Are there any general rules for designing such domains with similar functions to spider silk-derived NT*. Is spider silk-derived NT* unique? 5. In the title, "solubility domain" should be "soluble domain". The title is too big and should be tuned down since the whole study was done on a reporter protein.
Reviewer #3 (Remarks to the Author): In the manuscript "A spider silk-derived solubility domain inhibits nuclear and cytosolic protein aggregation in human cells", Schellhaus et al., studied the potential of a recombinant spidroinbased NT* domain in preventing protein aggregation in HeLa cells. In cells expressing recombinant NT* domain in fusion with an aggregation reporter, the soluble pool of the reporter increased and the propensity to aggregate was independent of the relative position of the NT* domain with respect to the aggregation reporter. The authors went on to conclude that the spidroin-derived NT* domain has a generic anti-aggregation activity, independent of position or subcellular location, that is also active in human cells and propose that the NT* domain can potentially be exploited in controlling protein aggregation of disease-associated proteins.
The study is interesting from a basic science perspective, builds over their previous work on the anti-aggregation potential of NT*, and together with earlier work, builds a strong rationale for testing the functions of such recombinant proteins in vivo. However, the current manuscript suffers from some issues, as outlined below, which renders it unsuitable for publication in its present form. This reviewer realizes the potential of the study and is willing to re-review a significantly revised manuscript for consideration in Communications Biology.

Major revision:
The physiological relevance of protein misfolding and aggregation has been studied most widely in the contexts of neurodegenerative disorders. Even in the references cited by the authors to introduce the generality of protein aggregation, the major focus has been the proteins related to amyloid or neurodegenerative disorders. In this context, this reviewer would feel more confident of the anti-aggregation potential of NT* in cells that are more relevant to such conditions than HeLa cells e.g., SH-SY5Y cells or any other relevant model. A didactic review in this regard was published in 2020 by Slanzi et al. (doi: 10.3389/fcell.2020.00328). This is because the secretory system for cellular trafficking of aggregation-prone proteins are differentially adapted in those specialized cell types than HeLa cells. The authors may use one such model to reproduce a subset of the results from HeLa cells to significantly improve the scientific appeal of the manuscript.
Minor revision: 1. The authors should revise the statement (line 119) that states " Administration of Shield1 did not further increase the solubility of AgDD-NT*-sfGFP suggesting that protein aggregation was efficiently prevented by insertion of the NT* domain." Fig.1c shows a quantifiable difference between the S and P panels in the absence or presence of S1.
2. All western blot experiments have been done on transiently transfected cells, whereas flow and microscopy on stable cell lines. It is important to repeat a subset of western blots on stable cell lines or provide reliable quality control data for transfection efficiency.

Reviewer 1
This authors have investigated the effect of the engineered spidroin NT* domain on induced aggregation within human cells. The domain has previously been developed and applied as a solubility and expression aid for partner proteins. Here they have combined the NT* domain with the small molecule Shield-1 FKBP ligand and have determined the effect of the NT* domain on modulation of aggregation, in the cytosol and nucleus of HeLa cells. This is a useful extension of previous work with the NT* domain and potentially opens up further avenues for use of the domain in biotechnology applications and as a tool for investigating the cellular impact of aggregation and rescue, with a variety of client proteins. It is likely to be of interest to many in the field.
The experimental results are of high quality and clearly described but there are some questions unanswered, regarding the generalisability of the system to other client proteins and impact of nuclear localisation. The inclusion of a disease-associated, aggregation-prone client protein for comparison with the inducible aggregation here, would greatly add to the impact of this work.
Authors: We are pleased that the reviewer appreciates the importance of our findings for the field. Below we discuss the concerns raised by the reviewer and how they have been addressed in the revised manuscript.
Points to be addressed: The authors should include a brief explanation of the nature of the two-residue modification that generates the NT* domain -i.e. explain specifics of residue charge switch. Likewise, brief description of the AgDD is important, nature of FKBP domain and Shield-1, should be included in the Introduction.

Authors: We have included in the Introduction section a better explanation of the NT* and AgDD domain and included additional citations. See lines 70-77 (NT*) and lines 87-89 (AgDD).
Authors should comment on the effect of position of the NT* domain on the levels of protein expression achieved. Line 129 Explain what is meant by "homogeneous expression of the transgene". How is this detected by flow cytometry? Is this referring to similar levels with different constructs, or cellular distribution, or lack of puncta? Authors: We agree that this statement was unclear but were referring to the fact that the expression levels within each clone were similar as a we obtained a Gaussian distribution indicative for a single population. We have replaced this statement with a more precise comment about the expression levels. See lines 133-138. Fig 2E show that the inclusion of the NT* domain leads to uniform, cytosolic distribution and no obvious GFP-positive puncta. However, Fig 2F shows that addition of Shield ligand leads to an apparently even distribution across nucleus and cytoplasm. Is there other evidence in the literature that the activity or response of the AgDD system is affected by nuclear or cytosolic localisation? The authors should include an NT*-GFP constructs as additional control to discriminate between effects of the ligand and NT* domain.

Authors: As the reviewer proposed, we have generated and tested an NT*-sfGFP construct. The NT*-sfGFP is equally distributed between the cytosolic and nuclear compartments in the absence or presence of ligand (Suppl. Fig. 1). This implies that the cytosolic localization in the absence of ligand of constructs containing both the NT* and AgDD domain can be attributed to the AgDD domain. This is also supported by the observation that the AgDD-sfGFP forms cytosolic aggregates but localizes to the nucleus in the presence of ligand, implying that the NT* domain is not required for this phenomenon. To the best of our knowledge, it has not been reported before that the Shield1 ligand affects the localization of AgDD fusion proteins. The AgDD fusions with the NT* domain may have made this feature more apparent as it keeps the fusion in a soluble state also in the absence of ligand. We discuss this in the Result section of the paper. See lines 159-162.
Additionally, the results presented Fig 3, where the NT* domain only provides ~50% rescue from aggregation in the absence of Shield-1 and inhibits rescue by Shield-1, require further investigation. Do the authors have other evidence that would report on the effect of an NLS on NT* activity and function? The authors should include NLS-NT*-GFP constructs or other data indicating that the NLS does not affect NT*.

Authors: Since the NT* does not have any effect in the context of an NLS-NT*-GFP fusion (as it is not aggregation prone), it would not be possible to test with the proposed construct whether the NLS affect the NT* domain. We do, however, show that the NT* domain prevents the aggregation of the NLS-AgDD-sfGFP-NT* in HeLa and DAOY cells, which demonstrates that the NLS domain does not interfere with this property of the NT* domain. Vice versa the NT* domain does not affect the NLS domain as this fusion still properly localizes to the nuclei in both cell lines.
Have the authors tested the effect of position of the NT* in these experiments, e.g. in NLS-AgDD-sfGFP-NT*?

Authors: We have not tested the NLS-AgDD-sfGFP-NT* domain but we have shown that 1) the NT* domain does not seem to interfere with the nuclear localization and 2) we have tested the functionality of the NT* domain in various positions for the AgDD-sfGFP fusions.
Please explain what is meant by "three independent experiments of the soluble and insoluble fractions"? Were fractions prepared from three independent biological replicates?
Authors: With three independent experiments, we meant that the entire procedure (seeding, transfection, harvesting) has been performed on separate occasions. We were not referring to replicate samples. We did not have the feeling that this description was ambiguous and open for different interpretations but tried to outline it even more explicit in the revision. See lines 363-365.

Reviewer #2
This work presented a study using spider silk-derived soluble domain to preventing protein aggregation in the human cell. Though the overall quality of the study is above average, some serious concerns have to be considered.  Biotechnol. 17, 353-358). This location ensures that the solubility tag is already present when the target protein is being produced and allows proteolytic removal of the tag without leaving protease recognition motifs in the target protein. Unpublished data from our lab show that recombinant production in bacteria of amyloid-beta peptide, islet amyloid polypeptide and nerve growth factor as target proteins give equally much soluble fusion protein with N-or Cterminally located NT* and similar data are published for other tags.
5. In the title, "solubility domain" should be "soluble domain". The title is too big and should be tuned down since the whole study was done on a reporter protein.
Authors: "Solubility domain" is a term that is general used for domains that increase the solubility of a protein. We feel that "soluble domain" can be misunderstood as it may be interpreted as a reference to the fact that the domain itself is soluble without indicating whether it affects the solubility of the entire protein.
Reviewer #3 (Remarks to the Author): In the manuscript "A spider silk-derived solubility domain inhibits nuclear and cytosolic protein aggregation in human cells", Schellhaus et al., studied the potential of a recombinant spidroin-based NT* domain in preventing protein aggregation in HeLa cells. In cells expressing recombinant NT* domain in fusion with an aggregation reporter, the soluble pool of the reporter increased and the propensity to aggregate was independent of the relative position of the NT* domain with respect to the aggregation reporter. The authors went on to conclude that the spidroin-derived NT* domain has a generic anti-aggregation activity, independent of position or subcellular location, that is also active in human cells and propose that the NT* domain can potentially be exploited in controlling protein aggregation of disease-associated proteins.
The study is interesting from a basic science perspective, builds over their previous work on the antiaggregation potential of NT*, and together with earlier work, builds a strong rationale for testing the functions of such recombinant proteins in vivo. However, the current manuscript suffers from some issues, as outlined below, which renders it unsuitable for publication in its present form. This reviewer realizes the potential of the study and is willing to re-review a significantly revised manuscript for consideration in Communications Biology.
Authors: We are pleased that the reviewer finds the study interesting and having potential. Below we outline the additional experimentation that has been done to address the concerns of the reviewer.

Major revision:
The physiological relevance of protein misfolding and aggregation has been studied most widely in the contexts of neurodegenerative disorders. Even in the references cited by the authors to introduce the generality of protein aggregation, the major focus has been the proteins related to