Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

Direct Outreach in Bars and Clubs to Enroll Cigarette Smokers in Mobile Cessation Services: Exploratory Study.

BACKGROUND: Cigarette smoking and alcohol use are well known to be concomitant behaviors, but there is a lack of studies related to recruitment of smokers for mobile cessation services at places where alcohol is consumed, such as bars and clubs. Adapting recruitment strategies to expand the reach of cessation programs to where tobacco users are located may help decrease the health-equity gap in tobacco control by improving reach and enrollment of underserved smokers residing in low-income and rural areas who are not reached by traditional cessation services. OBJECTIVE: The purpose of this exploratory study was to assess the feasibility of direct outreach in bars, clubs, and restaurants to recruit smokers to Quitxt, our mobile smoking cessation service. Quitxt is delivered through SMS text messaging or Facebook Messenger. METHODS: We collaborated with an advertising agency to conduct in-person recruitment of young adult smokers aged 18-29 years, focusing on urban and rural Spanish-speaking Latino participants, as well as English-speaking rural White and African American participants. Street team members were recruited and trained in a 4-hour session, including a brief introduction to the public health impacts of cigarette smoking and the aims of the project. The street teams made direct, face-to-face contact with smokers in and near smoking areas at 25 bars, clubs, and other venues frequented by young smokers in urban San Antonio and nearby rural areas. RESULTS: The 3923 interactions by the street teams produced 335 (8.5%) program enrollments. Most participants were English speakers with a mean age of 29.2 (SD 10.6) years and smoked a mean of 8.5 (SD 6.2) cigarettes per day. Among users who responded to questions on gender and ethnicity, 66% (70/106) were women and 56% (60/107) were Hispanic/Latino. Among users ready to make a quit attempt, 22% (17/77) reported 1 tobacco-free day and 16% (10/62) reported maintaining cessation to achieve 1 week without smoking. The response rate to later follow-up questions was low. CONCLUSIONS: Direct outreach in bars and clubs is a useful method for connecting young adult cigarette smokers with mobile cessation services. However, further research is needed to learn more about how mobile services can influence long-term smoking cessation among those recruited through direct outreach, as well as to test the use of incentives in obtaining more useful response rates.

Co-adaptor driven assembly of a CUL3 E3 ligase complex.

Cullin-RING E3 ligases (CRLs) are essential ubiquitylation enzymes that combine a catalytic core built around cullin scaffolds with ∼300 exchangeable substrate adaptors. To ensure robust signal transduction, cells must constantly form new CRLs by pairing substrate-bound adaptors with their cullins, but how this occurs at the right time and place is still poorly understood. Here, we show that formation of individual CRL complexes is a tightly regulated process. Using CUL3KLHL12 as a model, we found that its co-adaptor PEF1-ALG2 initiates CRL3 formation by releasing KLHL12 from an assembly inhibitor at the endoplasmic reticulum, before co-adaptor monoubiquitylation stabilizes the enzyme for substrate modification. As the co-adaptor also helps recruit substrates, its role in CRL assembly couples target recognition to ubiquitylation. We propose that regulators dedicated to specific CRLs, such as assembly inhibitors or co-adaptors, cooperate with target-agnostic adaptor exchange mechanisms to establish E3 ligase complexes that control metazoan development.

Facebook Chat Application to Prompt and Assist Smoking Cessation Among Spanish-Speaking Young Adults in South Texas.

Given how smart phones, internet services, and social media have shown great potential for assisting smoking cessation, we constructed a Facebook chat application based on our previous work with SMS texting services. This report summarizes findings from 2,364 Spanish-speaking young adults recruited through Facebook advertising in South Texas during the 2020 New Year holiday season. Among these service users, 926 (39%) were ready to make a quit attempt, and 26 (3.1%) of those users reported that they were tobacco free 1 month later. There were no responses to a chat question survey 72 days after the dates selected for quitting. Although more research with longer follow up is needed, these findings show that social media chat applications may be helpful for at least prompting quit attempts and short-term cessation among young adult Spanish-speaking smokers. There is no evidence of an impact on long-term cessation, and more research is clearly needed.

Structural basis for dimerization quality control.

Most quality control pathways target misfolded proteins to prevent toxic aggregation and neurodegeneration1. Dimerization quality control further improves proteostasis by eliminating complexes of aberrant composition2, but how it detects incorrect subunits remains unknown. Here we provide structural insight into target selection by SCF-FBXL17, a dimerization-quality-control E3 ligase that ubiquitylates and helps to degrade inactive heterodimers of BTB proteins while sparing functional homodimers. We find that SCF-FBXL17 disrupts aberrant BTB dimers that fail to stabilize an intermolecular ß-sheet around a highly divergent ß-strand of the BTB domain. Complex dissociation allows SCF-FBXL17 to wrap around a single BTB domain, resulting in robust ubiquitylation. SCF-FBXL17 therefore probes both shape and complementarity of BTB domains, a mechanism that is well suited to establish quality control of complex composition for recurrent interaction modules.

A Digital Health Intervention for Weight Management for Latino Families Living in Rural Communities: Perspectives and Lessons Learned During Development.

Rural residents face numerous challenges in accessing quality health care for management of chronic diseases (eg, obesity, diabetes), including scarcity of health care services and insufficient public transport. Digital health interventions, which include modalities such as internet, smartphones, and monitoring sensors, may help increase rural residents' access to health care. While digital health interventions have become an increasingly popular intervention strategy to address obesity, research examining the use of technological tools for obesity management among rural Latino populations is limited. In this paper, we share our experience developing a culturally tailored, interactive health intervention using digital technologies for a family-oriented, weight management program in a rural, primarily Latino community. We describe the formative research that guided the development of the intervention, discuss the process of developing the intervention technologies including issues of privacy and data security, examine the results of a pilot study, and share lessons learned. Our experience can help others design user-centered digital health interventions to engage underserved populations in the uptake of healthy lifestyle and disease management skills.

Facilitating the development of cross-platform mHealth applications for chronic supportive care and a case study.

Mobile health (mHealth) apps have received increasing attention, due to their abilities to support patients who suffer from various conditions. mHealth apps may be especially helpful for patients with chronic diseases, by providing pertinent information, tracking symptoms, and inspiring adherence to medication regimens. To achieve these objectives, researchers need to prototype mHealth apps with dedicated software architectures. In this paper, a cloud-based mHealth application development concept is presented for chronic patient supportive care apps. The concept integrates existing software platforms and services for simplified app development that can be reused for other target applications. This developmental method also facilitates app portability, through the use of common components found across multiple mobile platforms, and scalability, through the loose coupling of services. The results are demonstrated by the development of native Android and cross-platform web apps, in a case study that presents an mHealth solution for endocrine hormone therapy (EHT). A performance analysis methodology, an app usability evaluation, based on focus group responses, and alpha and pre-beta testing results are provided.

Technology-based health promotion: Current state and perspectives in emerging gig economy.

It has been a decade since smartphone application stores started allowing developers to post their own applications. This paper presents a narrative review on the state-of-the-art and the future of technology used by researchers in the field of mobile health promotion. Researchers build high cost, complex systems with the purpose of promoting health and collecting data. These systems promote health by using a feedback component that "educates" the subject. Other researchers instead use platforms which provide them with data collected by others, which allows for no communication with subjects, but may be cheaper than building a system to collect the data. This second type of systems cannot be used directly for health promotion. However, both types of systems are relevant to the field of health promotion, because they are precursors to a third type of systems that are emerging, the gig economy systems for mobile health data collection, which are low cost, globally available, and provide limited communication with subjects. If such systems evolve to include more channels for communication with the data-generating subjects, and also bring developers into the economy, they may eventually revolutionize the field of mobile health promotion and data collection by giving researchers new capabilities, such as the ability to replicate existing health promotion campaigns with the click of a button and the appropriate licenses. In this paper we present a review of state-of-the-art systems for mobile health promotion and data collection and a model for what these systems may look like in the future.

Principles of Ubiquitin-Dependent Signaling.

Ubiquitylation is an essential posttranslational modification that controls cell division, differentiation, and survival in all eukaryotes. By combining multiple E3 ligases (writers), ubiquitin-binding effectors (readers), and de-ubiquitylases (erasers) with functionally distinct ubiquitylation tags, the ubiquitin system constitutes a powerful signaling network that is employed in similar ways from yeast to humans. Here, we discuss conserved principles of ubiquitin-dependent signaling that illustrate how this posttranslational modification shapes intracellular signaling networks to establish robust development and homeostasis throughout the eukaryotic kingdom.

Text and Mobile Media Smoking Cessation Service for Young Adults in South Texas: Operation and Cost-Effectiveness Estimation.

To realize the promising potential of services delivered via smart phones to help young adults quit smoking at a high level of cost-efficiency, we constructed a texting and mobile media system that was promoted in South Texas via social media advertising and other recruitment channels. During the 6-month service period described here, enrollments were achieved for 798 participants with a mean age of 29.3 years. Seven-month texted follow-up found that 21% (171) of the enrollees reported abstinence at that point. This is consistent with high rates of success found in studies of telephone counseling for young adults and confirms that text and mobile media service specifically designed for young adults provide a feasible and potentially cost-effective approach to promoting cessation.

Conducting the finale of DNA replication.

Anomalies in dismantling the machinery of DNA replication can compromise genome integrity and contribute to tumorigenesis and aging. In this issue of Genes & Development, Dewar and colleagues (pp. 275-290) identified an E3 ubiquitin ligase, CUL2LRR2, that modifies a subunit of the replicative CMG (Cdc45, minichromosome maintenance [MCM] subunits 2-7, and the GINS complex) helicase and triggers disassembly of the replication machinery. Their study offers critical insight into the mechanism of DNA replication termination while at the same time raising important questions for future research.

Chemoproteomic Screening of Covalent Ligands Reveals UBA5 As a Novel Pancreatic Cancer Target.

Chemical genetic screening of small-molecule libraries has been a promising strategy for discovering unique and novel therapeutic compounds. However, identifying the targets of lead molecules that arise from these screens has remained a major bottleneck in understanding the mechanism of action of these compounds. Here, we have coupled the screening of a cysteine-reactive fragment-based covalent ligand library with an isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) chemoproteomic platform to rapidly couple the discovery of lead small molecules that impair pancreatic cancer pathogenicity with the identification of druggable hotspots for potential cancer therapy. Through this coupled approach, we have discovered a covalent ligand DKM 2-93 that impairs pancreatic cancer cell survival and in vivo tumor growth through covalently modifying the catalytic cysteine of the ubiquitin-like modifier activating enzyme 5 (UBA5), thereby inhibiting its activity as a protein that activates the ubiquitin-like protein UFM1 to UFMylate proteins. We show that UBA5 is a novel pancreatic cancer therapeutic target and show DKM 2-93 as a relatively selective lead inhibitor of UBA5. Our results underscore the utility of coupling the screening of covalent ligand libraries with isoTOP-ABPP platforms for mining the proteome for druggable hotspots for cancer therapy.

Regulation of the CUL3 Ubiquitin Ligase by a Calcium-Dependent Co-adaptor.

The ubiquitin ligase CUL3 is an essential regulator of neural crest specification whose aberrant activation has been linked to autism, schizophrenia, and hypertension. CUL3 exerts its roles by pairing with ∼90 distinct substrate adaptors, yet how the different CUL3-complexes are activated is poorly understood. Here, we show that CUL3 and its adaptor KLHL12 require two calcium-binding proteins, PEF1 and ALG2, for recognition of their substrate SEC31. PEF1 and ALG2 form a target-specific co-adaptor that translates a transient rise in cytosolic calcium levels into more persistent SEC31 ubiquitylation, which in turn triggers formation of large COPII coats and promotes collagen secretion. As calcium also instructs chondrocyte differentiation and collagen synthesis, calcium-dependent control of CUL3KLHL12 integrates collagen secretion into broader programs of craniofacial bone formation. Our work, therefore, identifies both calcium and CUL3 co-adaptors as important regulators of ubiquitylation events that control human development.

Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts.

ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. AIMS: The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. METHODOLOGY: Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor. ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. RESULTS: Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-α-phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone. Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone. CONCLUSION: Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.

Regulation of cargo recognition, commitment, and unloading drives cotranslational protein targeting.

Efficient and accurate protein localization is essential to cells and requires protein-targeting machineries to both effectively capture the cargo in the cytosol and productively unload the cargo at the membrane. To understand how these challenges are met, we followed the interaction of translating ribosomes during their targeting by the signal recognition particle (SRP) using a site-specific fluorescent probe in the nascent protein. We show that initial recruitment of SRP receptor (SR) selectively enhances the affinity of SRP for correct cargos, thus committing SRP-dependent substrates to the pathway. Real-time measurement of cargo transfer from the targeting to translocation machinery revealed multiple factors that drive this event, including GTPase rearrangement in the SRP-SR complex, stepwise displacement of SRP from the ribosome and signal sequence by SecYEG, and elongation of the nascent polypeptide. Our results elucidate how active and sequential regulation of the SRP-cargo interaction drives efficient and faithful protein targeting.

Assessing media access and use among Latina adolescents to inform development of a physical activity promotion intervention incorporating text messaging.

Increasing physical activity (PA) during preadolescence and adolescence is critical to reversing the obesity epidemic. A recent report described the promising role of eHealth--the use of new media for purposes of health promotion--in reducing and preventing childhood obesity. This study assessed access/use of various media (cell phones, computers, gaming systems, Internet) among adolescent Latino girls and examined the relationship between PA and media access/use. A convenience sample of 110 Latino girls ages 11 to 14 was recruited from Girl Scouts of Southwest Texas and other groups. The media survey was self-administered (April-July 2010) on personal digital assistants. Of the girls, 55% reported owning a cell phone and spending 40 (SD = 4.2) hours per week talking, texting, listening to music, and browsing the Internet. Cell phone access increased significantly with age (p = .029). Compared to those with no cell phone, girls with a cell phone have greater odds of reporting more than 5 days of PA in the past week (odds ratio = 5.5, 95% confidence interval [CI] = 2.1, 14) and engaging in daily physical education classes (odds ratio = 2.6, 95% CI = 1.1, 5.9). Since girls with cell phones report greater PA, cell phones may be an effective strategy for communicating with girls about engaging in PA.

SecYEG activates GTPases to drive the completion of cotranslational protein targeting.

Signal recognition particle (SRP) and its receptor (SR) comprise a highly conserved cellular machine that cotranslationally targets proteins to a protein-conducting channel, the bacterial SecYEG or eukaryotic Sec61p complex, at the target membrane. Whether SecYEG is a passive recipient of the translating ribosome or actively regulates this targeting machinery remains unclear. Here we show that SecYEG drives conformational changes in the cargo-loaded SRP-SR targeting complex that activate it for GTP hydrolysis and for handover of the translating ribosome. These results provide the first evidence that SecYEG actively drives the efficient delivery and unloading of translating ribosomes at the target membrane.

Signal recognition particle: an essential protein-targeting machine.

The signal recognition particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.

MessageSpace: a Messaging System for Health Research.

Mobile Health (mHealth) has emerged as a promising direction for delivery of healthcare services via mobile communication devices such as cell phones. Examples include texting-based interventions for chronic disease monitoring, diabetes management, control of hypertension, smoking cessation, monitoring medication adherence, appointment keeping and medical test result delivery; as well as improving patient-provider communication, health information communication, data collection and access to health records. While existing messaging systems very well support bulk messaging and some polling applications, they are not designed for data collection and processing of health research oriented studies. For that reason known studies based on text-messaging campaigns have been constrained in participant numbers. In order to empower healthcare promotion and education research, this paper presents a system dedicated for healthcare research. It is designed for convenient communication with various study groups, feedback collection and automated processing.