Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria.

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.

Development and pilot evaluation of Native CREST-a Cancer Research Experience and Student Training program for Navajo undergraduate students.

The Mayo Clinic Cancer Center and Diné College received funding for a 4-year collaborative P20 planning grant from the National Cancer Institute in 2006. The goal of the partnership was to increase Navajo undergraduates' interest in and commitment to biomedical coursework and careers, especially in cancer research. This paper describes the development, pilot testing, and evaluation of Native CREST (Cancer Research Experience and Student Training), a 10-week cancer research training program providing mentorship in a Mayo Clinic basic science or behavioral cancer research lab for Navajo undergraduate students. Seven Native American undergraduate students (five females, two males) were enrolled during the summers of 2008-2011. Students reported the program influenced their career goals and was valuable to their education and development. These efforts may increase the number of Native American career scientists developing and implementing cancer research, which will ultimately benefit the health of Native American people.

Vps9 domain-containing proteins: activators of Rab5 GTPases from yeast to neurons.

Endocytosis of cell surface receptors plays an important role in regulating cell signaling cascades. In some cases, internalization of an activated receptor attenuates the signaling process, while in other cases the clustering of activated receptors on early endosomal structures has been proposed to be essential for fully activating signaling cascades. Regulating the movement of receptors and other signaling proteins through the endocytic pathway, therefore, has a direct impact on cellular homeostasis. The small GTPase Rab5 is a crucial regulatory component of the endocytic pathway. Activation of Rab5 is mediated by GDP-GTP exchange factors (GEFs) that generate the Rab5-GTP complex. A large number of proteins have been identified that contain a specific, highly conserved domain (Vps9) that catalyzes nucleotide exchange on Rab5, linking the regulation of cell signaling cascades with intracellular receptor trafficking through the endocytic pathway.

Rapid Adaptation and Remote Delivery of Undergraduate Research Training during the COVID-19 Pandemic.

When COVID-19 caused worldwide cancellations of summer research immersion programs in 2020, Mayo Clinic rallied to create an alternate virtual experience called Summer Foundations in Research (SFIR). SFIR was designed not only to ensure the continuance of science pathways training for undergraduate scientists but also to support undergraduate mental wellbeing, given the known pandemic stressors. A total of 170 participants took part in the program and were surveyed pre-post for outcomes in biomedical research career knowledge, biomedical research career interest, research skills confidence, and three dimensions of mental wellbeing. Knowledge of and interest in careers involving biomedical research rose significantly following participation in SFIR. The participants' mean research skills confidence also rose between 0.08 and 1.32 points on a 7-point scale across 12 items from the Clinical Research Appraisal Inventory. Success in science pathways support was accompanied by positive shifts in participant mental wellbeing. Measurable decreases in stress (Perceived Stress Scale, p < 0.0001) accompanied gains in resilience (Brief Resilience Scale, p < 0.0001) and life satisfaction (Satisfaction with Life Scale, p = 0.0005). Collectively, the data suggest that core objectives of traditional in-person summer research programming can be accomplished virtually and that these programs can simultaneously impact student wellbeing. This theoretical framework is particularly salient during COVID-19, but the increased accessibility of virtual programs such as SFIR can continue to bolster science education pathways long after the pandemic is gone.

Rapid adaptation and remote delivery of undergraduate research training during the COVID 19 Pandemic.

COVID-19 continues to alter daily life around the globe. Education is particularly affected by shifts to distance learning. This change has poignant effects on all aspects of academic life, including the consequence of increased mental stress reported specifically for students. COVID-19 cancellations of many summer fellowships and internships for undergraduates across the country increased students' uncertainty about their educational opportunities and careers. When the pandemic necessitated elimination of on-campus programming at Mayo Clinic, a new program was developed for remote delivery. Summer Foundations in Research (SFIR) was drafted around 4 aims: 1) support the academic trajectory gap in research science created by COVID-19; 2) build sustainable scientific relationships with mentors, peers, and the community; 3) create opportunities for participants to share and address concerns with their own experiences in the pandemic; and 4) provide support for individual wellbeing. SFIR included research training, but also training in communication through generative Dialogue and resilience through Amit Sood's SMART program. 170 participants were followed for outcomes in these spaces. Knowledge of and interest in careers involving biomedical research rose significantly following SFIR. Participants' mean confidence levels in 12 Key areas of research rose between 0.08 to 1.32 points on a 7-point scale. The strongest gains in mean confidence levels were seen in designing a study and collaborating with others. SFIR participants demonstrated gains in perceived happiness, and measured resilience and a reduction in stress. Participants' qualitative responses indicated exceptionally positive mentor relationships and specific benefit of both the SMART program and Dialogue.

Cell proliferation and epidermal growth factor signaling in non-small cell lung adenocarcinoma cell lines are dependent on Rin1.

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Delta) complemented the Rin1 depletion effects, and overexpression of Rin1Delta had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.

Evaluating yeast biosynthetic vacuolar transport.

Study of the lysosomal protein transport system has been facilitated through dissection of the analogous vacuolar protein sorting (VPS) pathway in Saccharomyces cerevisiae. Resident enzymes of the yeast vacuole are synthesized as inactive precursors and are cleaved to their mature forms upon delivery to this compartment. Quantitative assessment of this delivery can be achieved through the use of pulse-chase experiments monitoring the cleavage of zymogens to their mature forms. The experimental procedures for analysis of carboxypeptidase Y (CPY) and carboxypeptidase S (CPS) maturation are described.

Biochemical characterization of Alsin, a Rab5 and Rac1 guanine nucleotide exchange factor.

Alsin is the gene product mutated in three juvenile-onset neurodegenerative disorders including amyotrophic lateral sclerosis 2 (ALS2). Sequence motif searches within Alsin predict the presence of Vps9, DH, and PH domains, implying that Alsin may function as a guanine nucleotide exchange factor (GEF) for Rab5 and a member of the Rho GTPase family. Procedures are presented in this chapter for the expression, purification, and biochemical characterization of the individual GEF domains of Alsin. A fractionation method is also described for the determination of Alsin's subcellular distribution. The presence of both Rac1 and Rab5 GEF activities makes Alsin a unique dual exchange factor that may couple endocytosis (via Rab5 activation) to cytoskeletal modulation (via Rac1 activation).

Ubiquitin regulation of the Rab5 family GEF Vps9p.

To maintain cellular homeostasis, the levels of transmembrane receptors found on the plasma membrane must be tightly regulated. Endocytosis of activated receptors and the eventual degradation of these transmembrane proteins in the lysosome serve a vital role in maintaining the plasma membrane receptor levels as well as attenuating the downstream signaling pathways. Two processes that regulate this receptor trafficking are the covalent modification of the receptor with ubiquitin (ubiquitylation) and the activation of the Rab5 family of small GTPases. Activation of Rab5 family proteins has been shown to be critical for early steps of the endocytic pathway including delivery of activated receptors to the early endosome, while ubiquitylation of activated receptors has been shown to be involved in receptor internalization, delivery to the endosome, and sorting into the multivesiclar body. In yeast, the guanine nucleotide exchange factor Vps9p serves to integrate the activation of a Rab5 protein (Vps21p) via the Vps9 domain with ubiquitin binding via the CUE domain to facilitate the delivery of ubiquitylated receptors to the endosome. Here we provide detailed protocols for the study of Vps9p in vivo and in vitro with regard to Vps21p activation, ubiquitin binding, and Vps9p ubiquitylation.

Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly.

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

Cross-species characterization of the ALS2 gene and analysis of its pattern of expression in development and adulthood.

Mutations in the ALS2 gene, which encodes alsin, cause autosomal recessive juvenile-onset amyotrophic lateral sclerosis (ALS2) and related conditions. Using both a novel monoclonal antibody and LacZ knock-in mice, we demonstrate that alsin is widely expressed in neurons of the CNS, including the cortex, brain stem and motor neurons of the spinal cord. Interestingly, the highest levels of alsin are found in the molecular layer of the cerebellum, a brain region not previously implicated in ALS2. During development, alsin is expressed by day E9.5, but CNS expression does not become predominant until early postnatal life. At the subcellular level, alsin is tightly associated with endosomal membranes and is likely to be part of a large protein complex that may include the actin cytoskeleton. ALS2 is present in primates, rodents, fish and flies, but not in the nematode worm or yeast, and is more highly conserved than expected among mammals. Additionally, the product of a second, widely expressed gene, ALS2 C-terminal like (ALS2CL), may subserve or modulate some of the functions of alsin as an activator of Rab and Rho GTPases.

Alsin is a Rab5 and Rac1 guanine nucleotide exchange factor.

ALS2 is the gene mutated in a recessive juvenile form of amyotrophic lateral sclerosis (ALS2). ALS2 encodes a large protein termed alsin, which contains a number of predicted cell signaling and protein trafficking sequence motifs. To gain insight into the overall function of alsin and to begin to evaluate its role in motor neuron maintenance, we examined the subcellular localization of alsin and the biochemical activities associated with its individual subdomains. We found that the Vps9p domain of alsin has Rab5 guanine nucleotide exchange activity. In addition, alsin interacted specifically with and acted as a guanine nucleotide exchange factor for Rac1. Immunofluorescence and fractionation experiments in both fibroblasts and neurons revealed that alsin is a cytosolic protein, with a significant portion associated with small, punctate membrane structures. Many of these membrane structures also contained Rab5 or Rac1. Upon overexpression of full-length alsin, the overexpressed material was largely cytosolic, indicating that the association with membrane structures could be saturated. We also found that alsin was present in membrane ruffles and lamellipodia. These data suggest that alsin is involved in membrane transport events, potentially linking endocytic processes and actin cytoskeleton remodeling.

The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase.

The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

Mechanism of ubiquitin recognition by the CUE domain of Vps9p.

Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways. The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain. The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule. The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin. The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule. Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.

Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic.

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.