Microbiomes for All.

Microbiome research projects are often interdisciplinary, involving fields such as microbiology, genetics, ecology, evolution, bioinformatics, and statistics. These research projects can be an excellent fit for undergraduate courses ranging from introductory biology labs to upper-level capstone courses. Microbiome research projects can attract the interest of students majoring in health and medical sciences, environmental sciences, and agriculture, and there are meaningful ties to real-world issues relating to human health, climate change, and environmental sustainability and resilience in pristine, fragile ecosystems to bustling urban centers. In this review, we will discuss the potential of microbiome research integrated into classes using a number of different modalities. Our experience scaling-up and implementing microbiome projects at a range of institutions across the US has provided us with insight and strategies for what works well and how to diminish common hurdles that are encountered when implementing undergraduate microbiome research projects. We will discuss how course-based microbiome research can be leveraged to help faculty make advances in their own research and professional development and the resources that are available to support faculty interested in integrating microbiome research into their courses.

One-Year Research Experience for Associate's Degree Students Impacts Graduation, STEM Retention, and Transfer Patterns.

The CUNY Research Scholars Program (CRSP) provides a yearlong faculty-mentored research experience to associate's degree students. The program takes place at all 10 associate's degree-granting colleges within the City University of New York system. We report on a mixed-methods study of 500 students who participated in the program during its initial 3 years. Quantitative longitudinal assessments revealed that students who engaged in CRSP were more likely to be retained in a science, technology, engineering, and mathematics (STEM) discipline or to graduate with a STEM degree than their counterparts in a matched comparison group. Furthermore, students who participated in CRSP demonstrated an increased likelihood of transferring to the more research-intensive 4-year schools within the CUNY system and to R1 universities outside the CUNY system. CRSP students reported an increased sense of belonging in college based on survey data, and focus groups with their mentors provided insight into the factors that led to the gains listed above. These combined results-of student data analysis, student surveys, and mentor focus groups-provide evidence that early research experiences for associate's degree students contribute to their academic success.

Specificity in the actions of the UBR1 ubiquitin ligase in the degradation of nuclear receptors.

The UBR1 ubiquitin ligase promotes degradation of proteins via the N-end rule and by another mechanism that detects a misfolded conformation. Although UBR1 was shown recently to act on protein kinases whose misfolding was promoted by inhibition of Hsp90, it was unknown whether this ubiquitin ligase targeted other client types of the chaperone. We analyzed the role of UBR1 in the degradation of nuclear receptors that are classical clients of Hsp90. Our results showed that UBR1 deletion results in impaired degradation of the glucocorticoid receptor and the androgen receptor but not the estrogen receptor α. These findings demonstrate specificity in the actions of the UBR1 ubiquitin ligase in the degradation of Hsp90 clients in the presence of small molecule inhibitors that promote client misfolding.

A network of ubiquitin ligases is important for the dynamics of misfolded protein aggregates in yeast.

Quality control ubiquitin ligases promote degradation of misfolded proteins by the proteasome. If the capacity of the ubiquitin/proteasome system is exceeded, then misfolded proteins accumulate in aggregates that are cleared by the autophagic system. To identify components of the ubiquitin/proteasome system that protect against aggregation, we analyzed a GFP-tagged protein kinase, Ste11ΔN(K444R)-GFP, in yeast strains deleted for 14 different ubiquitin ligases. We show that deletion of almost all of these ligases affected the proteostatic balance in untreated cells such that Ste11ΔN(K444R)-GFP aggregation was changed significantly compared with the levels found in wild type cells. By contrast, aggregation was increased significantly in only six E3 deletion strains when Ste11ΔN(K444R)-GFP folding was impaired due to inhibition of the molecular chaperone Hsp90 with geldanamycin. The increase in aggregation of Ste11ΔN(K444R)-GFP due to deletion of UBR1 and UFD4 was partially suppressed by deletion of UBR2 due to up-regulation of Rpn4, which controls proteasome activity. Deletion of UBR1 in combination with LTN1, UFD4, or DOA10 led to a marked hypersensitivity to azetidine 2-carboxylic acid, suggesting some redundancy in the networks of quality control ubiquitin ligases. Finally, we show that Ubr1 promotes clearance of protein aggregates when the autophagic system is inactivated. These results provide insight into the mechanics by which ubiquitin ligases cooperate and provide feedback regulation in the clearance of misfolded proteins.

UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition.

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.

Quality control and fate determination of Hsp90 client proteins.

Quality control processes regulate the proteome by determining whether a protein is to be folded or degraded. Hsp90 is a hub in the network of molecular chaperones that maintain this process because it promotes both folding and degradation, in addition to regulating expression of other quality control components. The significance of Hsp90's role in quality control is enhanced by the function of its clients, which include protein kinases and transcription factors, in cellular signaling. The inhibition of Hsp90 with small molecules results in the rapid degradation of such clients via the ubiquitin/proteasome pathway, and also in the induction of the Hsp70 molecular chaperone. These two events result in markedly different outcomes depending on cell type. For tumor cells there is a profound loss of signaling in growth promoting pathways. By contrast, increased amounts of Hsp70 in neuronal cells ameliorate the toxicity that is associated with the formation of aggregates observed in neurodegenerative conditions. In this review we discuss the mechanisms underlying these differential effects of Hsp90 inhibition on the quality control of distinct client proteins. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

Role of molecular chaperones in biogenesis of the protein kinome.

Molecular chaperones promote polypeptide folding in cells by protecting newly made and otherwise misfolded proteins against aggregation or degradation by the ubiquitin proteasome pathway. The roles of Saccharomyces cerevisiae Cdc37 and Ydj1 molecular chaperones are described in this chapter. We focus on biogenesis of protein kinases that require several different molecular chaperones for their proper folding. Specific among these is Cdc37, which binds directly to its kinase clients either during or shortly after translation and protects them against rapid proteasomal degradation. Ydj1 has a similar role, but is less specific for protein kinases in its role as a molecular chaperone. The method that we describe uses pulse chase and immunoprecipitation to analyze the fate of newly made proteins. Two kinetically distinct pathways of degradation can be discerned using this methodology that is dependent on the presence of an Hsp90 inhibitor or occurs in mutants of the molecular chaperones under study. The first is "zero-point" degradation that occurs either during or immediately after translation. The second is a slower pathway, where the half-life of kinase is approximately 20 min after translation.

Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins.

Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.

Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

Ydj1 protects nascent protein kinases from degradation and controls the rate of their maturation.

Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Delta mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Delta mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Delta mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation.

Multiple kinases and system robustness: a link between Cdc37 and genome integrity.

We identified 38 genes as having a genetic interaction with a mutant form of the kinase specific chaperone, Cdc37, using a genome-wide synthetic screening approach. The identified genes included a sub-network of highly interacting genes enriched for functions in genome integrity and comprising multiple components of several discrete molecular machines. A network analysis approach related these machines to a small group of cell cycle checkpoint kinases.

Akt shows variable sensitivity to an Hsp90 inhibitor depending on cell context.

Hsp90 inhibitors are currently in clinical trials for cancer therapy based on their ability to promote proteasomal degradation of oncogenic protein kinases and nuclear receptors. Results from recent studies suggest that cancer cells are more sensitive to these inhibitors than cells from healthy tissues. We analyzed an immortalized cell line Ba/F3 for sensitivity to the Hsp90 inhibitor geldanamycin in the absence and presence of the oncogenic tyrosine fusion kinase NPM-ALK expressed from a retroviral vector. Our results showed that NPM-ALK expression makes Akt and Cdk4 more resistant to degradation in the presence of geldanamycin, and there was a slightly reduced amount of apoptosis. The mechanism underlying the effect of NPM-ALK on Akt stability was probed by comparison of the turnover of the kinase after translation inhibition and geldanamycin treatment. We observed that Akt was degraded more rapidly in the presence of GA than upon translation inhibition without NPM-ALK expression. This suggests that NPM-ALK protects the mature kinase. Furthermore, Akt failed to bind to the Cdc37 chaperone in cells expressing NPM-ALK, which also correlates with increased Akt stability.

Uncoupling of hormone-dependence from chaperone-dependence in the L701H mutation of the androgen receptor.

The mechanisms underlying androgen receptor (AR)-mediated progression of prostate cancer following androgen ablation have yet to be fully determined. On this basis we screened naturally occurring mutants of human AR for hormone-independent activity using a yeast model system. An initial screen of 43 different mutants revealed that ARs having a Leu701His mutation (AR(L701H)) exhibited hormone-independent activation of a lacZ reporter gene. The AR(L701H) mutant bound dihydrotestosterone to a similar extent as did wild type AR, although its ability to be induced by hormone for transactivation was reduced substantially. Subsequent studies focused on the dependence of AR(L701H) on molecular chaperones for folding to the active state. We found that AR(L701H) was highly dependent on Hsp90 for its hormone-independent activation, suggesting that this chaperone functions in AR(L701H) folding. However, the mutant did not respond specifically to increased levels of FKBP52, suggesting that this chaperone functions at the hormone-dependent activation stage in the folding process. Further studies of AR(L701H) in PC3 cells suggested that this mutant is prohibited from hormone-independent transactivation in mammalian cells. However, basal expression of a reporter gene by AR(L701H) was not impaired by the presence of 17-allylamino-17-demethoxygeldanamycin as was wild type AR, suggesting differential interactions of these receptors with molecular chaperones in animal cells.

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation.

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.

Molecular chaperones and protein kinase quality control.

The Hsp90-Cdc37 chaperone pair has special responsibility for folding of protein kinases. This function has made Hsp90 a target for new chemotherapeutic approaches, and several compounds are currently being tested for their ability to inhibit many different kinases simultaneously. Not all kinases are sensitive to these inhibitors, however, and this difference might depend on how each kinase interacts with Hsp90 and Cdc37 during folding of the nascent chain and thereafter. Indeed, several kinases require the persistent presence of both chaperones after initial folding and some of these kinases seem to be particularly sensitive to Hsp90 inhibitors. This requirement might relate to conformational changes that take place during the protein kinase activity cycle.

Differential effects of the organochlorine pesticide DDT and its metabolite p,p'-DDE on p-glycoprotein activity and expression.

1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) is an organochlorine pesticide. Its metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethene (p,p'-DDE) is a persistent environmental contaminant and both compounds accumulate in animals. Because multidrug resistance transporters, such as p-glycoprotein, function as a defense against xenobiotic exposure, we analyzed the ability of DDT and p,p'-DDE to act as efflux modulators. Using a competitive intact cell assay based on the efflux of the fluorescent dye rhodamine 123, we found that DDT, but not p,p'-DDE, stimulated dye retention. Subsequent studies using verapamil as competitor suggested that DDT is a weak p-glycoprotein inhibitor. Further studies addressed the ability of DDT and p,p'-DDE to induce MDR1, the gene encoding p-glycoprotein. In HepG2 cells, we found that both compounds induced MDR1 by twofold to threefold. Similar results were observed in mouse liver after a single dose of p,p'-DDE, although some gender-specific induction differences were noted. By contrast, p,p'-DDE failed to induce MDR1 in HeLa cells, indicating some cell-specific effects for induction. Further expression studies demonstrated increased levels of the endoplasmic reticulum molecular chaperone, Bip, in response to DDT, but not p,p'-DDE. These results suggest that DDT, but not p,p'-DDE, induces an endoplasmic reticulum stress response.

The type I Hsp40 zinc finger-like region is required for Hsp70 to capture non-native polypeptides from Ydj1.

The cytosolic yeast Hsp40 Ydj1 contains a conserved zinc finger-like region (ZFLR), which has two zinc-binding domains (ZBD), that helps regulate and specify Hsp70 function. To investigate the mechanism for Ydj1 ZFLR action, ZBDI and ZBDII mutants were constructed and characterized. ZBDII mutants exhibited temperature-sensitive growth defects, but yeast tolerated mutation of ZBDI. However, ZBDI and ZBDII mutants were defective at facilitating androgen receptor (AR) folding. Defective AR folding was associated with the accumulation of complexes between AR and Ydj1 ZFLR mutants and a reduction in Hsp70.AR complex formation. Purified Ydj1 ZBDI and ZBDII mutants could bind non-native polypeptides but could not deliver luciferase to Hsp70 and were defective at luciferase refolding. Interestingly, the ability of Ydj1 to synergize with Hsp70 to suppress thermally induced protein aggregation was blocked by mutation of ZBDII, but not ZBDI. Hence, ZBDII is required for yeast to survive heat stress because it is essential for Ydj1 to cooperate with Hsp70 to suppress protein aggregation. On the other hand, protein folding is dependent upon the action of both ZBDI and ZBDII because each is required for Hsp70 to capture non-native polypeptides from Ydj1.

Oxandrolone blocks glucocorticoid signaling in an androgen receptor-dependent manner.

The anabolic steroid oxandrolone is increasingly used to preserve or restore muscle mass in those with HIV infection or serious burns. These effects are mediated, in part, by the androgen receptor (AR). Anti-glucocorticoid effects have also been reported for some anabolic steroids, and the goal of our studies was to determine whether oxandrolone had a similar mechanism of action. Studies with in vitro translated glucocorticoid receptor (GR), however, showed no inhibition of cortisol binding by oxandrolone. Conversely, experiments in cell culture systems demonstrated significant antagonism of cortisol-induced transcriptional activation by oxandrolone in cells expressing both the AR and GR. Inhibition was not overcome by increased cortisol concentration, and no inhibition by oxandrolone was observed in cells expressing GR alone, confirming that non-competitive mechanisms were involved. AR-dependent repression of transcriptional activation by oxandrolone was also observed with the synthetic glucocorticoids dexamethasone and methylprednisolone. Furthermore, the AR antagonists 2-hydroxyflutamide and DDE also repressed GR transactivation in an AR-dependent manner. A mutant AR lacking a functional nuclear localization signal (AR(4RKM)) was active in oxandrolone-mediated repression of GR even though oxandrolone-bound AR(4RKM) failed to enter the nucleus and did not affect nuclear import of GR. These data indicate a novel action of oxandrolone to suppress glucocorticoid action via crosstalk between AR and GR.

Sti1 and Cdc37 can stabilize Hsp90 in chaperone complexes with a protein kinase.

Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown. We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase. Studies on signaling via this protein kinase pathway was measured by alpha-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1. Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents. The greatest defects were in cpr7Delta, sse1Delta, and ydj1Delta mutants. Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies. Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Delta mutant back to wild-type levels. CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Delta mutant strain. These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes. Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.

Identification of a conserved sequence motif that promotes Cdc37 and cyclin D1 binding to Cdk4.

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4(G15A) and Cdk4(G18A) were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4(G15A) and Cdk4(G18A) relative to wild type Cdk4, suggesting that Cdk4(G15A) and Cdk4(G18A) adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4(G15A), and Cdk4(G18A) had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4(G15A). Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.