Biology subjects

Dutta, N.

Publications and source records attributed to Dutta, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Expression of tardigrade intrinsically disordered proteins in C. elegans reveals variable effects on aging and stress

Tardigrades survive extreme environmental stress by expressing intrinsically disordered proteins that stabilize cellular structures during cryptobiosis. Because these proteins protect cells from desiccation, oxidative stress, and DNA damage, they have attracted interest as potential therapeutics to enhance stress resilience in other organisms. However, most studies have relied on cultured cells or germline expression, leaving their effects in somatic tissues largely unexplored. Here, we generated Caenorhabditis elegans strains expressing four tardigrade intrinsically disordered proteins, Damage Suppressor (DSUP), cytoplasmic-abundant heat-soluble (CAHS), mitochondrial-abundant heat-soluble (MAHS), and Ramazzottius varieornatus late embryogenesis abundant (RvLEAM), throughout somatic tissues. Surprisingly, constitutive expression of each protein reduced lifespan, impaired motility and reproduction, and failed to improve resistance to most environmental stressors. DSUP toxicity was tissue- and developmental stage-dependent, indicating that both spatial and temporal expression influence its physiological effects. Despite these detrimental phenotypes, several proteins reduced protein aggregation during desiccation-associated stress, demonstrating context-dependent protective effects. Together, our findings reveal that tardigrade stress proteins impose physiological tradeoffs in vivo and underscore the importance of evaluating candidate stress-protective proteins in physiologically relevant somatic models before therapeutic translation.

genetics↗

bioRxiv2026

elDORS: An elevated Database Of RNA Sequences

Massive and integrated sequence databases have revolutionized computational protein structure prediction. RNA lags due to a lack of consolidated sequence resources. To bridge this gap, we developed elDORS_raw, which comprises up-to-date sequence information, including recent metagenomes and transcriptome sequence data. We optimized an 80% sequence-identity clustered version named elDORS for use with the RNAcmap3 split-strategy for multiple sequence alignment (MSA) and the widely used rMSA pipeline. Our benchmarking demonstrates that elDORS-augmented MSA pipelines match or exceed the alignment depth obtained with massive legacy databases across different queries, including blind CASP challenges, effectively eliminating sequence retrieval failures challenging orphan RNAs. To aid homology searches, predicting RNA properties, training new models, and other downstream tasks, elDORS is freely accessible. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/737016v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@d7549aorg.highwire.dtl.DTLVardef@f36ab1org.highwire.dtl.DTLVardef@e1a07eorg.highwire.dtl.DTLVardef@efdab4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Cryo-EM structures of anti Z-DNA antibodies in complex with antigen reveal distinct recognition modes of a left-handed geometry

Double-stranded nucleic acids can undergo transitions from canonical B/A-forms to alternate left-handed Z-DNA/Z-RNA (Z-NAs). Z-NAs are implicated in processes such as neuroinflammation in Alzheimers disease, Lupus Erythematosus, microbial biofilms, and type I interferon-mediated human pathologies. Since endogenous Z-NA sensors like the Z domain can induce B-to-Z transitions, monoclonal antibodies (mAbs) Z-D11 and Z22 have been regarded as conformation-specific tools to confirm Z-NA in situ, although high-resolution structural information is missing. Here, we employed single-particle cryo-electron microscopy to solve structures of Z-D11 and Z22 bound to synthetic d(CG)6 12mer Z-DNA duplex. Both mAbs form filamentous trimers around the Z-DNA axis, further stabilized by Fab-Fab interactions. The mAbs achieve specificity through extensive contacts to both Z-form backbone strands and the exposed guanine/cytosine bases in the major groove. This mode of recognition is dictated by shape complementarity rather than sequence specificity, sensing the alternating syn/anti backbone torsions and the phosphate zig-zag geometry unique to Z-DNA. Our data also suggest that these mAbs are not inducing B-to-Z transitions under normal physiological conditions. Finally, comparison to other double-stranded NA-binding mAbs defines a similar structural logic adapted to different helical geometry recognition patterns, thus providing a framework for engineering highly specific nucleic acid probes.

biochemistry↗

bioRxiv2025

Epoxyazadiradione ameliorates Parkinson's disease by upregulating heat shock factor 1 and protein degradation pathways in mice.

Parkinsons disease (PD) is a major debilitating health concern for millions of the elderly population all over the world. This progressive neurodegenerative disorder also poses a severe mental and financial burden to caregivers and society. Despite a major thrust on research for therapy development, no significant progress has been made; only temporary management options are currently available. To this end, we have reported azadiradione (AZD), a triterpenoid that we isolated from neem seed extract using a cell-based assay. AZD showed high efficacy in ameliorating protein aggregation-induced pathology and symptoms in fruit flies and mice. Current evidence suggests that AZD functions through activating the transcriptional function of heat shock factor 1 (HSF1), a master regulator of protein quality control pathways, without modulating the cellular redox balance. To better understand the pharmacophore of AZD, a triterpenoid in its observed function, we have analysed various structural derivatives, focusing on their HSF1-activating function in vitro and their efficacies in ameliorating protein aggregation-induced toxicities in cell and mouse models. Our analyses, based on real-time PCR, immunoblots, fluorescent anisotropy, and a mouse model of MPTP-induced PD, highlighted Epoxy-azadiradione (Epoxy) as being as efficient as AZD in in vivo functional tests, albeit activating the promoter binding activity of HSF1 with at least two-fold higher efficacy in vitro. Notably, similar to AZD, Epoxy did not induce cellular redox imbalance. We also incorporated molecular docking analyses involving the published crystal structure of the DNA-binding domain of HSF1 bound to its DNA recognition element to study molecular dynamics-based energy estimation. The analysis revealed a higher energy stability of the epoxy-bound complexes, as indicated by a significant decrease in binding free energy ({Delta}G) estimated from an ensemble of intermediate docked complex structures.

pharmacology and toxicology↗

bioRxiv2025

Form and function of actin impacts actin health and aging.

The actin cytoskeleton is a fundamental and highly conserved structure that functions in diverse cellular processes, yet its direct contribution to organismal aging remains unclear. Here, we systematically interrogated how genetic and pharmacologic perturbations of actin structure and function influence lifespan and various hallmarks of aging in Caenorhabditis elegans. Whole-animal and tissue-specific knockdown of actin and key actin-binding proteins (ABPs) - arx-2 (Arp2/3), unc-60 (cofilin), and lev-11 (tropomyosin) - led to premature disruption of filament organization, reduced lifespan, and tissue-specific physiological defects. Bulk and single-nucleus RNA-sequencing revealed that ABP knockdowns elicited a strongly "aged" transcriptome. Actin dysfunction broadly exacerbated many age-associated phenotypes, including mitochondrial dysfunction, lipid dysregulation, loss of proteostasis, impaired autophagy, and intestinal barrier failure. Pharmacological destabilization with Latrunculin A mirrored genetic knockdowns, while mild stabilization with Jasplakinolide modestly extended lifespan, emphasizing that optimal and finely-tuned actin function is critical for healthy aging. Finally, analysis of human genome-wide association data revealed that common ACTB polymorphisms correlate with differences in age-related decline in gait speed, suggesting evolutionary conservation of actins role in healthy aging. Taken together, our results provide a comprehensive and publicly accessible resource that maps, for the first time, how actin integrity intersects with diverse aging pathways across tissues and scales. This descriptive framework is intended to enable future mechanistic discovery by offering a deep, unbiased dataset that can be integrated with emerging studies to define how actin dynamics contribute to aging.

cell biology↗

bioRxiv2025

Transcriptomic analysis of mitohormesis associated with lifespan extension in Caenorhabditis elegans

Non-lethal exposure to mitochondrial stress has been shown to have beneficial effects due to activation of signalling pathways, including the mitochondrial unfolded protein response (UPRmt). Activation of UPRmt restores function of the mitochondria and improves general health and longevity in multiple model systems, termed mitohormesis. In C. elegans, mitohormesis can be accomplished by electron transport chain inhibition, decline in mitochondrial translation, decreased mitochondrial import, and numerous other methods that activate UPRmt. However, not all methods that activate UPRmt can promote longevity. These and other studies have started to question whether UPRMT is directly correlated with longevity. Here, we attempt to address this controversy by unravelling the complex molecular regulation of longevity of the nematode under different mitochondrial stressors that induce UPRmt by performing RNA-sequencing to profile transcriptome changes. Using this comprehensive and unbiased approach, we aim to determine whether specific transcriptomic changes can reveal a correlation between UPRmt and longevity. Altogether this study will provide mechanistic insights on mitohormesis and how it correlates with lifespan of C. elegans.

systems biology↗

bioRxiv2025

HSF1 Activator Azadiradione Ameliorates Parkinson Disease and Extends Lifespan in Preclinical Models: Analysis of Underlying Molecular Mechanism

Parkinsons disease (PD) affects millions worldwide, with no efficient therapy currently available. A major cause of the initiation and progression of this degenerative disease is the dysfunctional cellular protein quality control system (PQC), leading to the accumulation of toxic protein aggregates in neurons. We previously reported azadiradione (AZD), a small molecule (MW 451 Da), as a potent inducer of heat shock factor 1 (HSF1) activity, which could alleviate cellular toxicity induced by misfolded proteins by upregulating the levels of inducible molecular chaperones and proteasome activity. Here, we show the multifaceted effect of AZD in enhancing the capacity of PQC machinery in cells, fruit flies, and a PD mouse model. AZD activated HSF1 by promoting its phosphorylation at S326 through MEK. In parallel, AZD boosted protein degradation through increased chymotrypsin-like proteasome activity, upregulation of the ubiquitin ligase CHIP. AZD induced autophagy, marked by elevated levels of Beclin 1, ATG7, and ULK1 phosphorylation at S555, along with mTORC1 inhibition via AMPK activation. Surprisingly, the calorie restriction pathway was also upregulated upon AZD treatment, as demonstrated by the enhanced phosphorylation of FOXO3 and FOXO1, along with increased activity of their target enzymes SOD and catalase. Notably, AKT activity was also suppressed in AZD-treated cells. In vivo, AZD improved motor function, dopaminergic neuron survival, and tyrosine hydroxylase activity in an MPTP-induced mouse model of PD, and extended lifespan in Drosophila without compromising fertility or mobility. These findings highlight AZD as a promising therapeutic candidate for restoring PQC and mitigating PD pathology.

molecular biology↗

bioRxiv2025

RNAhub - an automated pipeline to search and align RNA homologs with secondary structure assessment

The complexity in the generation of RNA multiple sequence alignments and assessment of the accuracy of such alignments contributes to the challenges in the utilization of RNA alignments in diverse integrative methods. RNAhub is a freely available user-friendly web server for a reliable generation of RNA multiple sequence alignments and the detection of the presence of structural RNA utilizing evolutionary information. This web-based tool, developed by the integration of existing computational approaches, takes an RNA sequence as input and automatically retrieves and aligns sequences homologous to the input (query) RNA sequence through an iterative and structure-agnostic approach. Based on the alignment, this tool statistically assesses whether the query RNA sequence has a conserved RNA structure using covariation analysis. The web server allows the user to efficiently search the sequence of interest against carefully curated, ready-to-use genomic databases to produce a multiple sequence alignment. Using this alignment, our tool either detects the presence of a conserved structural RNA, finds evidence against the presence of a conserved structure, or cannot make any assessment due to a lack of sequence diversity in the alignment. The web server is freely available at https://rnahub.org. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=181 HEIGHT=200 SRC="FIGDIR/small/642701v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@540f6forg.highwire.dtl.DTLVardef@c15e53org.highwire.dtl.DTLVardef@117ced2org.highwire.dtl.DTLVardef@b679c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Cross comparison of imaging strategies of mitochondria in C. elegans during aging.

Mitochondria are double membrane-bound organelles with pleiotropic roles in the cell, including energy production through aerobic respiration, calcium signaling, metabolism, proliferation, immune signaling, and apoptosis. Dysfunction of mitochondria is associated with numerous physiological consequences and drives various diseases, and is one of twelve biological hallmarks of aging, linked to aging pathology. There are many distinct changes that occur to the mitochondria during aging including changes in mitochondrial morphology, which can be used as a robust and simple readout of mitochondrial quality and function. Although mitochondrial morphology alone cannot be used to conclude the quality of mitochondria, it is highly correlated with mitochondrial function whereby mitochondria exhibit increased fragmentation with age in multiple cell types of the nematode C. elegans. Thus, C. elegans serve as a robust model for rapidly measuring mitochondrial morphology changes during aging. To standardize imaging methods for mitochondrial morphology in C. elegans, we provide a detailed comparative characterization of several transgenic constructs, highlighting benefits and caveats for aging biology studies. Summary BlurbThis study evaluates mitochondrial imaging in C. elegans during aging, comparing various transgenic constructs for tissue-specific mitochondrial visualization. The findings highlight technical considerations, imaging method standardization, and the utility of C. elegans as a robust model for studying mitochondrial dynamics.

cell biology↗

bioRxiv2024

Structural and dynamic consequences of inosine, 2'-O-methylation, and N6-methyladenosine modifications in RNA G-quadruplex: A molecular dynamics study

RNA G-quadruplexes (rG4) are stable non-canonical structures, often found to regulate important biological processes such as transcription, translation, splicing, RNA localization, and other steps in gene expression. rG4 structures can also influence DNA-related processes e.g. DNA replication, telomere elongation and homeostasis, and recombination. Due to the involvement of rG4s in such important processes, these structures are potential therapeutic targets against different diseases e.g., viral infection and cancer. Hence, a better understanding of the structure and stability of rG4s and their role in various therapeutically important cellular processes can help in the design of effective therapeutic strategies for targeting such structures. In the present study, we present our findings on the structural and dynamic effect of RNA modifications (inosine; 2'-O-methylation, and m6A-methylation) on RNA G-quadruplex structure from molecular dynamics simulations. Additionally, we also report the dynamic and energetic aspects of inter-quadruplex interactions and the influence of non-G purine tetrads neighboring the inter-quadruplex junction on the interactions.

biophysics↗

bioRxiv2024

Distinct mechanisms of non-autonomous UPRER mediated by GABAergic, glutamatergic, and octopaminergic neurons.

The capacity to deal with stress declines during the aging process, and preservation of cellular stress responses is critical to healthy aging. The unfolded protein response of the endoplasmic reticulum (UPRER) is one such conserved mechanism, which is critical for the maintenance of several major functions of the ER during stress, including protein folding and lipid metabolism. Hyperactivation of the UPRER by overexpression of the major transcription factor, xbp-1s, solely in neurons drives lifespan extension as neurons send a neurotransmitter-based signal to other tissue to activate UPRER in a non-autonomous fashion. Previous work identified serotonergic, dopaminergic, and tyraminergic neurons in this signaling paradigm. To further expand our understanding of the neural circuitry that underlies the non-autonomous signaling of ER stress, we activated UPRER solely in glutamatergic, octopaminergic, and GABAergic neurons in C. elegans and paired whole-body transcriptomic analysis with functional assays. We found that UPRER-induced signals from glutamatergic neurons increased expression of canonical protein homeostasis pathways and octopaminergic neurons promoted pathogen response pathways; while minor, statistically significant changes were observed in lipid metabolism-related genes with GABAergic UPRER activation. These findings provide further evidence for the distinct role neuronal subtypes play in driving the diverse response to ER stress.

cell biology↗

bioRxiv2024

Mechanical stress through growth on stiffer substrates impacts animal health and longevity in C. elegans.

Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo, model organism setting to study the impact of mechanical stress on aging, by increasing substrate stiffness in solid agar medium of C. elegans. To our surprise, we found shockingly limited impact of growth of C. elegans on stiffer substrates, including limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our studies reveal that altering substrate stiffness of growth medium for C. elegans have only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.

cell biology↗

bioRxiv2023

Investigating impacts of marine sponge derived mycothiazole and its acetylated derivative on mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), a widely used ETC complex I inhibitor, these two molecules showed cytotoxicity to cancer cells but strikingly demonstrate a lack of toxicity to non-cancer cells, a highly beneficial feature in the development of anti-cancer therapeutics. Furthermore, in vivo experiments with these small molecules utilizing C.elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. Interestingly, we also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilize the transcription factors ATFS-1 and HSF-1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF-1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

cell biology↗

bioRxiv2023

Glial-derived mitochondrial signals impact neuronal proteostasis and aging.

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPRMT), between tissues. However, these studies largely ignored non-neuronal cells of the nervous system. Here, we found that UPRMT activation in four, astrocyte-like glial cells in the nematode, C. elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Surprisingly, we find that glial cells utilize small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then impact neuron-mediated effects in organismal homeostasis and longevity. One-Sentence SummaryGlial cells sense mitochondrial stress and signal a beneficial stress signal to promote neuronal health and longevity.

genetics↗

bioRxiv2023

Structural and thermodynamic consequences of base pairs containing pseudouridine and N1-methylpseudouridine in RNA duplexes

Pseudouridine ({Psi}) is one of the most common post-transcriptional modifications in RNA and has been known to play significant roles in several crucial biological processes. The N1-methyl derivative of pseudouridine i.e N1-methylpseudouridine has also been reported to be important for the stability and function of RNA. Several studies suggest the importance of pseudouridine and N1-methylpseudouridine in mRNA therapeutics. The critical contribution of pseudouridine, especially that of its N1-methyl derivative in the efficiency of the COVID-19 mRNA vaccines, suggests the requirement to better understand the role of these modifications in the structure, stability and function of RNA. In the present study, we have investigated the consequences of the presence of these modifications in the stability of RNA duplex structures by analyzing different structural properties, hydration characteristics and energetics of these duplexes. We have previously studied the structural and thermodynamic properties of RNA duplexes with an internal {Psi}-A pair and reported the stabilizing effect of {Psi} over U (Deb, I. et al. Sci Rep 9, 16278 (2019)). Here, we have extended our work to understand the properties of RNA duplexes with an internal m1{Psi}-A pair and also theoretically demonstrate the effect of substituting internal U-G, U-U and U-C mismatches with the {Psi}-G, {Psi}-U and {Psi}-C mismatches and also with the m1{Psi}-G, m1{Psi}-U and m1{Psi}-C mismatches respectively, within dsRNA. Our results indicate the context-dependent stabilization of base stacking interactions by N1-methylpseudouridine compared to uridine and pseudouridine, presumably resulting from the increased molecular polarizability due to the presence of the methyl group.

biophysics↗

bioRxiv2023

EnCPdock: a web-interface for direct conjoint comparative analyses of complementarity and binding energetics in inter-protein associations

ContextProtein-protein interaction (PPI) is a key component linked to virtually all cellular processes. Be it an enzyme catalysis ( classic type functions of proteins) or a signal transduction ( non-classic), proteins generally function involving stable or quasi-stable multi-protein associations. The physical basis for such associations is inherent in the combined effect of shape and electrostatic complementarities (Sc, EC) of the interacting protein partners at their interface, which provides indirect probabilistic estimates of the stability and affinity of the interaction. While Sc is a necessary criterion for inter-protein associations, EC can be favorable as well as disfavored (e.g., in transient interactions). Estimating equilibrium thermodynamic parameters ({Delta}Gbinding, Kd) by experimental means is costly and time consuming, thereby opening windows for computational structural interventions. Attempts to empirically probe {Delta}Gbinding from coarse-grain structural descriptors (primarily, surface area based terms) have lately been overtaken by physics-based, knowledge-based and their hybrid approaches (MM/PBSA, FoldX etc.) that directly compute {Delta}Gbinding without involving intermediate structural descriptors. MethodsHere we present EnCPdock (www.scinetmol.in/EnCPdock/), a user-friendly web-interface for the direct conjoint comparative analyses of complementarity and binding energetics in proteins. EnCPdock returns an AI-predicted {Delta}Gbinding computed by combining complementarity (Sc, EC) and other high-level structural descriptors (input feature vectors), and, renders a prediction accuracy comparable to the state-of-the-art. EnCPdock further locates a PPI complex in terms of its {Sc, EC} values (taken as an ordered pair) in the two-dimensional Complementarity Plot (CP). In addition, it also generates mobile molecular graphics of the interfacial atomic contact network for further analyses. EnCPdock also furnishes individual feature trends along with the relative probability estimates (Prfmax) of the obtained feature-scores with respect to the events of their highest observed frequencies. Together, these functionalities are of real practical use for structural tinkering and intervention as might be relevant in the design of targeted protein-interfaces. Combining all its features and applications, EnCPdock presents a unique online tool that should be beneficial to structural biologists and researchers across related fraternities.

bioinformatics↗

bioRxiv2022

Large-scale genetic screens identify BET-1 as a cytoskeleton regulator promoting actin health and lifespan.

The actin cytoskeleton is a three-dimensional scaffold of proteins that is a regulatory, energy-consuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well understood. Here, we performed a multi-pronged, cross-organismal screen combining a whole-genome CRISPR-Cas9 screen in human fibroblasts with in vivo C. elegans synthetic lethality screening. We identified the bromodomain protein, BET-1, as a key regulator of actin health and longevity. Overexpression of bet-1 preserves actin health at late age and promotes lifespan and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET-1. Together, our discovery assigns a key role for BET-1 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.

cell biology↗

bioRxiv2019

Collateral fitness effects of mutations

The distribution of fitness effects (DFE) of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutations collateral fitness effects, which we define as effects that do not derive from changes in the proteins ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the E. coli TEM-1 {beta}-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins, collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper post-translational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly-expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against mutations with deleterious effects on protein activity and protein abundance. Significance StatementMutations provide the source of genetic variability upon which evolution acts. Deleterious protein mutations are commonly thought of in terms of how they compromise the proteins ability to perform its physiological function. However, mutations might also be deleterious if they cause negative effects on one of the countless other cellular processes. The frequency and magnitude of such collateral fitness effects is unknown. Our systematic study of mutations in a bacterial protein finds widespread collateral fitness effects that were associated with protein aggregation, improper protein processing, incomplete protein transport across membranes, incorrect disulfide-bond formation, induction of stress-response pathways, and unexpected changes in cell properties. Our results suggest that deleterious collateral fitness effects may be an important constraint on protein evolution.

evolutionary biology↗