CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression.

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1-tomato PTO immune complex via acetylation.

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues.

Loss of a 7q gene, CUX1, disrupts epigenetically driven DNA repair and drives therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.

Biomarkers of Pediatric Cataracts: A Proteomics Analysis of Aqueous Fluid.

Cataracts are among the most common causes of childhood vision loss worldwide. This study seeks to identify differentially expressed proteins in the aqueous humor of pediatric cataract patients. Samples of aqueous humor were collected from pediatric and adult cataract patients and subjected to mass spectrometry-based proteomic analysis. Samples of pediatric cataracts were grouped by subtype and compared to adult samples. Differentially expressed proteins in each subtype were identified. Gene ontology analysis was performed using WikiPaths for each cataract subtype. Seven pediatric patients and ten adult patients were included in the study. Of the pediatric samples, all seven (100%) were male, three (43%) had traumatic cataracts, two (29%) had congenital cataracts, and two (29%) had posterior polar cataracts. Of the adult patients, seven (70%) were female and seven (70%) had predominantly nuclear sclerotic cataracts. A total of 128 proteins were upregulated in the pediatric samples, and 127 proteins were upregulated in the adult samples, with 75 proteins shared by both groups. Gene ontology analysis identified inflammatory and oxidative stress pathways as upregulated in pediatric cataracts. Inflammatory and oxidative stress mechanisms may be involved in pediatric cataract formation and warrant further investigation.

Simple strategies to enhance discovery of acetylation post-translational modifications by quadrupole-orbitrap LC-MS/MS.

Enzyme-dependent post-translational modifications (PTMs) mediate the cellular regulation of proteins and can be discovered using proteomics. However, even where the peptides of interest can be enriched for analysis with state-of-the-art LC-MS/MS tools and informatics, only a fraction of peptide ions can be identified confidently. Thus, many PTM sites remain undiscovered and unconfirmed. In this minireview, we use a case study to discuss how the use of inclusion lists, turning off isotopic exclusion, and manual validation significantly increased depth of coverage, facilitating discovery of acetylation sites in targets of an acetyltransferase virulence factor. These underutilized strategies have the potential to help answer many mechanistic biological questions that large-scale proteomic studies cannot.

A method for whole protein isolation from human cranial bone.

The presence of the dense hydroxyapatite matrix within human bone limits the applicability of conventional protocols for protein extraction. This has hindered the complete and accurate characterization of the human bone proteome thus far, leaving many bone-related disorders poorly understood. We sought to refine an existing method of protein extraction from mouse bone to extract whole proteins of varying molecular weights from human cranial bone. Whole protein was extracted from human cranial suture by mechanically processing samples using a method that limits protein degradation by minimizing heat introduction to proteins. The presence of whole protein was confirmed by western blotting. Mass spectrometry was used to sequence peptides and identify isolated proteins. The data have been deposited to the ProteomeXchange with identifier PXD003215. Extracted proteins were characterized as both intra- and extracellular and had molecular weights ranging from 9.4 to 629 kDa. High correlation scores among suture protein spectral counts support the reproducibility of the method. Ontology analytics revealed proteins of myriad functions including mediators of metabolic processes and cell organelles. These results demonstrate a reproducible method for isolation of whole protein from human cranial bone, representing a large range of molecular weights, origins and functions.

CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex.

CUX1 is a homeodomain-containing transcription factor that is essential for the development and differentiation of multiple tissues. CUX1 is recurrently mutated or deleted in cancer, particularly in myeloid malignancies. However, the mechanism by which CUX1 regulates gene expression and differentiation remains poorly understood, creating a barrier to understanding the tumor-suppressive functions of CUX1. Here, we demonstrate that CUX1 directs the BAF chromatin remodeling complex to DNA to increase chromatin accessibility in hematopoietic cells. CUX1 preferentially regulates lineage-specific enhancers, and CUX1 target genes are predictive of cell fate in vivo. These data indicate that CUX1 regulates hematopoietic lineage commitment and homeostasis via pioneer factor activity, and CUX1 deficiency disrupts these processes in stem and progenitor cells, facilitating transformation.

Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells.

BACKGROUND: Immune tolerance contributes to resistance to conventional cancer therapies such as radiation. Radiotherapy induces immunogenic cell death, releasing a burst of tumor antigens, but this appears insufficient to stimulate an effective antitumor immune response. Radiation also increases infiltration of cytotoxic T lymphocytes (CTLs), but their effector function is short lived. Although CTL exhaustion may be at fault, combining immune checkpoint blockade with radiation is insufficient to restore CTL function in most patients. An alternative model is that antigen presentation is the limiting factor, suggesting a defect in dendritic cell (DC) function. METHODS: Building on our prior work showing that cancer cells treated with radiation in the presence of the poly(ADP-ribose) polymerase-1 inhibitor veliparib undergo immunogenic senescence, we reexamined senescent cells (SnCs) as preventative or therapeutic cancer vaccines. SnCs formed in vitro were cocultured with splenocytes and evaluated by scRNA-seq to examine immunogenicity. Immature bone-marrow-derived DCs cocultured with SnCs were examined for maturation and activation by flow cytometry and T cell proliferation assays. Viable SnCs or SnC-activated DCs were injected subcutaneously, and vaccine effects were evaluated by analysis of immune response, prevention of tumor engraftment, regression of established tumors and/or potentiation of immunotherapy or radiotherapy. RESULTS: Murine CT26 colon carcinoma or 4T1 mammary carcinoma cells treated with radiation and veliparib form SnCs that promote DC maturation and activation in vitro, leading to efficient, STING-dependent CTL priming. Injecting mice with SnCs induces antigen-specific CTLs and confers protection from tumor engraftment. Injecting immunogenic SnCs into tumor-bearing mice increases inflammation with activated CTLs, suppresses tumor growth, potentiates checkpoint blockade, enhances radiotherapy and blocks colonization by disseminated tumor cells. Addressing the concern that reinjecting tumor cells into patients may be impractical, DCs activated with SnCs in vitro were similarly effective to SnCs in suppressing established tumors and blocking metastases. CONCLUSIONS: Therapeutic vaccines based on senescent tumor cells and/or SnC-activated DCs have the potential to improve genotoxic and immune therapies and limit recurrence or metastasis.

Proteomic analysis defines the interactome of telomerase in the protozoan parasite, Trypanosoma brucei.

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.

Genomic studies controvert the existence of the CUX1 p75 isoform.

CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110, generated by an alternative transcriptional start site or post-translational cleavage, respectively. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform as previously described. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization.

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Epidermal stratification requires retromer-mediated desmoglein-1 recycling.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Rapid validation of Mascot search results via stable isotope labeling, pair picking, and deconvolution of fragmentation patterns.

Conventional LC-MS/MS data analysis matches each precursor ion and fragmentation pattern to their best fit within databases of theoretical spectra, yielding a peptide identification. Confidence is estimated by a score but can be validated by statistics, false discovery rates, and/or manual validation. A weakness is that each ion is evaluated independently, discarding potentially useful cross-correlations. In a classical approach to de novo sequence analysis, mixtures of peptides differing only in a carboxyl-terminal isotopic label yield fragmentation spectra with single, unlabeled b-type ions but pairs of isotope-labeled y-type ions, facilitating confident assignments. To apply this principle to identification by fragmentation pattern matching, we developed Validator, software that recognizes isotopic peptide pairs and compares their identifications and fragmentation patterns. Testing Validator 1 on a Mascot results file from FT-ICR LC-MS/MS of (16)O/(18)O-labeled yeast cell lysate peptides yielded 2,775 peptide pairs sharing a common identification but differing in carboxyl-terminal label. Comparing observed b- and y-ions with the predicted fragmentation pattern improved the threshold Mascot score for 5% false discovery from 36 to 22, significantly increasing both sensitivity and specificity. Validator 2, which identifies pairs by precursor mass difference alone before comparing observed fragmentation with that predicted by Mascot, found 2,021 isotopic pairs, similarly achieving improved sensitivity and specificity. Finally Validator 3, which finds pairs based on mass difference alone and then deconvolutes fragmentation patterns independently of Mascot, found 964 predicted peptides. Validator 3 allowed raw mass spectrometry data to be mined not only to validate Mascot results but also to discover peptides missed by Mascot. Using standard desktop hardware, the Validator 1-3 software processed the 11,536 spectra in the 93-MB Mascot .DAT file in less than 6 min (32 spectra/s), revealing high confidence peptide identifications without regard to Mascot score, far faster than manual or other independent validation methods.

Lipid-derived electrophiles mediate the effects of chemotherapeutic topoisomerase I poisons.

Topoisomerase 1 (Top1) reversibly nicks chromosomal DNA to relax strain accumulated during transcription, replication, chromatin assembly, and chromosome condensation. The Top1 poison camptothecin targets cancer cells by trapping the enzyme in the covalent complex Top1cc, tethered to cleaved DNA by a tyrosine-3'-phosphate bond. In vitro mechanistic studies point to interfacial inhibition, where camptothecin binding to the Top1-DNA interface stabilizes Top1cc. Here we present a complementary covalent mechanism that is critical in vivo. We observed that camptothecins induce oxidative stress, leading to lipid peroxidation, lipid-derived electrophile accumulation, and Top1 poisoning via covalent modification. The electrophile 4-hydroxy-2-nonenal can induce Top1cc on its own and forms a Michael adduct to a cysteine thiol in the Top1 active site, potentially blocking tyrosine dephosphorylation and 3' DNA phosphate release. Thereby, camptothecins may leverage a physiological cysteine-based redox switch in Top1 to mediate their selective toxicity to rapidly proliferating cancer cells.

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Innate-like self-reactive B cells infiltrate human renal allografts during transplant rejection.

Intrarenal B cells in human renal allografts indicate transplant recipients with a poor prognosis, but how these cells contribute to rejection is unclear. Here we show using single-cell RNA sequencing that intrarenal class-switched B cells have an innate cell transcriptional state resembling mouse peritoneal B1 or B-innate (Bin) cells. Antibodies generated by Bin cells do not bind donor-specific antigens nor are they enriched for reactivity to ubiquitously expressed self-antigens. Rather, Bin cells frequently express antibodies reactive with either renal-specific or inflammation-associated antigens. Furthermore, local antigens can drive Bin cell proliferation and differentiation into plasma cells expressing self-reactive antibodies. These data show a mechanism of human inflammation in which a breach in organ-restricted tolerance by infiltrating innate-like B cells drives local tissue destruction.

Host expression system modulates recombinant Hsp70 activity through post-translational modifications.

The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.

Genetic analysis of Hsp70 phosphorylation sites reveals a role in Candida albicans cell and colony morphogenesis.

Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8 were only detected in the hyphal cells. Genetic analysis of these sites revealed allele-specific effects on growth or hyphae formation at 42 °C. Colony morphology, which is normally wrinkled or crenellated at 37 °C, reverted to smooth in several mutants, but this colony morphology phenotype was unrelated to cellular morphology. Two mutants exhibited a mild increase in sensitivity to the cell wall-active compounds caspofungin and calcofluor white. We suggest that this analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence. The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client proteins, and that modification of these sites can play cellular regulatory roles, consistent with the "chaperone code" hypothesis.

Dataset of Nematostella vectensis Hsp70 isoform interactomes upon heat shock.

Nematostella vectensis is an estuarine sea anemone that has emerged as a model species to characterize molecular responses to physiological stressors due to its exposure to diverse, extreme abiotic conditions. In marine cnidarians, Hsp70 proteins can be effective biomarkers to determine mechanisms of physiological acclimation and evolutionary adaptations to environmental stress: a pressing issue as concerns about climate change grow. Here we show the results of affinity purification mass spectrometry of three Nematostella vectensis Hsp70 isoforms, NvHsp70A, B and D when expressed in untreated and heat shocked yeast cells lacking their native Hsp70s. We identified a total of 1031 interactors for the three NvHsp70 isoforms, 549 or which were shared. NvHsp70 isoform interactions altered substantially under heat stress with 17% of NvHsp70A, 51% of NvHsp70B and 20% of NvHsp70D interactions increasing after exposure to 39 °C for 2 hours. For further interpretation of the data presented in this article, please see the research article "Dynamic remodeling of the interactomes of Nematostella vectensis Hsp70 isoforms under heat shock".

Dynamic remodeling of the interactomes of Nematostella vectensis Hsp70 isoforms under heat shock.

Heat shock protein 70s (Hsp70s) are a highly conserved class of molecular chaperones that fold a large proportion of the proteome. Nematostella vectensis (Nv) is an estuarine sea anemone that has emerged as a model species to characterize molecular responses to physiological stressors due to its exposure to diverse, extreme abiotic conditions. Previous transcriptional data has shown dramatic differences among expression profiles of three NvHsp70 isoforms (NvHsp70A, B and D) under stress but it is unknown if, and to what extent, the client proteins for these chaperones differ. In order to determine client specificity, NvHsp70A, B and D were expressed in Saccharomyces cerevisiae budding yeast lacking native Hsp70 and interacting proteins for each Hsp70 were determined with mass spectrometry in yeast ambient and heat shock conditions. Our analyses showed <50% of identified interacting proteins were common to all three anemone Hsp70s and 3-18% were unique to an individual Hsp70. Mapping of temperature induced interactions suggest that under stress a proportion of clients are transferred from NvHsp70A and NvHsp70D to NvHsp70B. Together, these data suggest a diverse set of interacting proteins for Hsp70 isoforms that likely determines the precise functions for Hsp70s in organismal acclimation and potentially adaptation. BIOLOGICAL SIGNIFICANCE: Although the Hsp70 family of molecular chaperones has been studied for >50 years, it is still not fully understood why organisms encode and express many highly-similar Hsp70 isoforms. The prevailing theory is that these isoforms have identical function, but are expressed under unique cellular conditions that include heat shock to cope with increased number of unfolded/misfolded proteins. The sea anemone Nematostella vectensis encodes three Hsp70 isoforms A, B and D that when expressed in yeast demonstrate unique functionalities. This study provides the interactome of NvHsp70s A, B and D and demonstrates that Hsp70 isoforms, while highly similar in sequence, have unique co-chaperone and client interactors.