ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors.

During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.

Development of an OTUD1 ubiquitin variant inhibitor.

OTUD1 (Ovarian tumor domain-containing deubiquitinase 1) is a member of the OTU domain-containing deubiquitinase family of enzymes involved in immunoregulation and tumorigenesis pathways. OTUD1 consists of three distinct regions: an unstructured N-terminal region, an OTU-fold catalytic domain, and a ubiquitin-interacting motif (UIM) containing region. Enhanced enzymatic activity and a strong preference for K63-linked substrates are imparted by the UIM containing region. We used phage display with a ubiquitin variant (UbV) library to identify binders for OTUD1 lacking the unstructured N-terminal region (OTUD1OTU + UIM) in an attempt to identify inhibitors bridging the catalytic domain and the UIM containing region. Two UbVs were identified (UbVOD.1 and UbVOD.2) with high affinity and specificity for OTUD1. Of the UbVs identified, UbVOD.1 inhibited OTUD1 activity towards mono-Ub and K63-linked di-Ub substrates in vitro with single-digit nanomolar IC50 and potently inhibited deubiquitinase activity with poly-Ub chains of other linkages. In vivo expression of UbVOD.1 alone was unstable, however as a di-UbV, global deubiquitination and deubiquitinase activity with the OTUD1 substrate RIPK1 were inhibited. Herein we describe the development of molecular tools for exploring the activity of OTUD1 in a cellular context, towards protein-based therapeutics.

"All Patients Are Not Treated as Equal": Extending Medicine's Social Contract to Black/African American Communities.

PHENOMENON: The social contract is an implicit agreement that governs medicine's values, beliefs, and practices in ways that uphold the profession's commitment to society. While this agreement is assumed to include all patients, historical examples of medical experimentation and mistreatment suggest that medicine's social contract has not been extended to Black patients. We suggest that is because underlying medicine's contract with society is another contract; the racial contract, which favors white individuals and legitimizes the mistreatment of those who are nonwhite. When Black/African American physicians enter medicine, they enter into the social contract as an agreement with society, but must navigate the realities of the racial contract in ways that have yet to be acknowledged. This study examines how Black/African American physicians interpret and enact the social contract in light of the country's racial contract by investigating the ways in which Black/African American physicians discuss their interactions with Black patients. APPROACH: This qualitative study reexamines cross-sectional data previously collected in 2018-2019 examining the professional identity formation (PIF) experiences of Black/African American trainees and physicians in the Southern part of the U.S. The goal of the larger study was to explore participants' professional identity formation experiences as racialized individuals within a predominantly white profession. The current study examines these data in light of medicine's social contract with society and Mill's (1997) theory of the racial contract to understand how Black physicians interpret and enact the social contract. Participants included 10 Black/African American students, eight residents, and nine attending physicians. FINDINGS: The findings show that Black/African American physicians and trainees are aware of the country's racial contract, which has resulted in Black patients being historically excluded from what has been described in the social contract that governs all physicians. As such, they are actively working to extend the social contract so that it includes Black patients and their communities. Specifically, they engage in trust building with the Black community to make sure all patients are included. Building trust includes ensuring a consistent stream of new Black/African American trainees, and equipping Black trainees and patients with the skills needed to improve the healthcare within the Black/African American community. INSIGHTS: While it been has assumed that all patients are included in the social contract between medicine and society, historical examples of medical mistreatment and experimentation demonstrate this is inaccurate; Black/African American communities have not been included. In an effort to dismantle systemic racism in the U.S., medical education must teach about its racist past and divulge how some communities have been historically excluded, providing new ways to think about how to include everyone in medicine's social contract.

"Changing the narrative": a study on professional identity formation among Black/African American physicians in the U.S.

Professional identity formation (PIF) is considered a key process in physician development. However, early PIF research may have inadvertently left out experiences from ethnically/racially minoritized physicians. As a result, the PIF literature may have forwarded dominant perspectives and assumptions about PIF that does not reflect those of minoritized physicians. This study used a cross-sectional study design, in which interview data was initially collected using constructivist grounded theory and then analyzed using critical lenses. Participants included 14 Black/African American students, 10 residents, and 17 attending physicians at two Southern medical schools in the U.S. Coding included the both/and conceptual framework developed out of Black feminist scholarship, and further analyzed using medicine's culture of Whiteness. These lenses identified assumptions made in the dominant PIF literature and how they compared to the experiences described by Black physicians. The results show that medical education's historical exclusion of minoritized physicians in medical education afforded a culture of Whiteness to proliferate, an influence that continues to frame the PIF research. Black physicians described their professional identity in terms of being in service to their racial/ethnic community, and the interconnectedness between personal/professional identities and context. Their professional identity was used to challenge larger social, historical, and cultural mistreatment of Black Americans, findings not described in the dominant PIF research. Black physicians' experiences as minoritized individuals within a culture of Whiteness reveals that the PIF literature is limited, and the current framings of PIF may be inadequate to study minoritized physicians.

Identification of ANKRD13D as a potential target in renal cell carcinomas.

BACKGROUND: The correlation of the expression of ankyrin repeat domain (ANKRD) family members with renal cell carcinoma prognosis was investigated. METHODS: The GEPIA2, GEO2R, UALCAN, GDC, OncoLnc, TIMER, PanglaoDB, CancerSEA, and Tabula Muris databases were used. Twelve ANKRD family members were identified as having overexpressed renal cell carcinoma samples. The ANKRD13D was identified as a renal cell carcinoma-specific target by cross-referencing the multiple survival databases. To clarify the role of ANKRD13D, the expression of NAKRD13D was analyzed at the single-cell level. RESULTS: ANKRD13D was mainly expressed in immune cells and positively correlated with Treg cell infiltration. The expression of ANKRD13D was also positively correlated with PDCD1, CTLA4, LAG3, TNFSF14, and ISG20. The overexpression of ANKRD13D in Treg was confirmed using reverse transcription-quantitative polymerase chain reaction. The structure of ANKRD13D was predicted using AlphaFold. CONCLUSION: In conclusion, we identified ANKRD13D as a key immune regulator, and targeting ANKRD13D with immune checkpoints blockade may be a promoting strategy for renal cell carcinoma immunotherapy.

Structural mechanisms underlying signaling in the cellular response to DNA double strand breaks.

DNA double strand breaks (DSBs) constitute one of the most dangerous forms of DNA damage. In actively replicating cells, these breaks are first recognized by specialized proteins that initiate a signal transduction cascade that modulates the cell cycle and results in the repair of the breaks by homologous recombination (HR). Protein signaling in response to double strand breaks involves phosphorylation and ubiquitination of chromatin and a variety of associated proteins. Here we review the emerging structural principles that underlie how post-translational protein modifications control protein signaling that emanates from these DNA lesions.

Perceived Barriers to Increasing Diversity within Oculofacial Plastic Surgery.

Purpose Physician diversity is limited in ophthalmology and oculofacial plastic surgery. Determination of barriers within the application process for oculofacial plastic surgery may help target efforts to improve the recruitment of underrepresented groups. This study aimed to illuminate perceived barriers to increasing diversity in oculofacial plastic surgery trainees, according to the American Society of Ophthalmic Plastic and Reconstructive Surgery (ASOPRS) fellows and fellowship program directors (FPDs). Methods During the month of February 2021, we sent surveys out to 54 current oculofacial plastic surgery fellows and 56 FPDs at 56 oculofacial plastic surgery programs recognized by the ASOPRS nationwide using a 15-question Qualtrics survey. Results Sixty-three individuals (57%) responded to the survey: 34 fellows (63%) and 29 FPDs (52%). Eighty-eight percent of fellows and 68% of FPDs identified as non-underrepresented in medicine (UiM). Forty-four percent of fellows and 25% of FPDs identified as men. FPDs most commonly noted, "Not enough minorities applying to our program" and "The objective data (Ophthalmic Knowledge Assessment Program score, United States Medical Licensing Examination Step scores, clinical honors, Alpha Omega Alpha status, letter of recommendation) for minority applicants often do not meet the threshold required to offer an interview or to be ranked to match" as barriers. Among fellows, the lowest-rated considerations when applying to oculofacial plastic surgery were "Racially/ethnically diverse faculty" and "Perceptions of minority candidates by fellowship programs," whereas "Likelihood of matching in program of choice" was ranked highest in considerations. Fellows identifying as men indicated greater concern for "Financial factors related to fellowship (e.g., loans, salary, cost of living, or cost of interviewing)" compared to fellows identifying as women who noted greater concern for "Program or preceptor acceptance of starting or having a family during fellowship." Conclusion Responses from FPDs suggest that efforts focused on recruiting and supporting diverse students to medicine and ophthalmology, mentoring applicants interested in oculofacial plastic surgery, and restructuring the application process to decrease bias, may improve diversity within the subspecialty. The lack of UiM representation in this study, 6% fellows and 7.4% FPDs identified as UiM, shows both the stark underrepresentation and the need for further research into this topic.

For us by us: Instituting mentorship models that credit minoritized medical faculty expertise and lived experience.

The woefully low proportion of scientists and clinicians underrepresented in medicine (UIM), including members of African-American/Black, Hispanic/Latinx, American Indian/Alaska Native or Native Hawaiian/Pacific Islander communities, is well characterized and documented. Diversity in medicine is not only just, but it improves quality and outcomes. Yet, diversity in academic medicine remains stagnant, despite national recognition and urgent calls to improve diversity, equity, and inclusion across health sciences. One strategy that has shown to improve diversity in many sectors is high quality mentoring. While many institutions have adopted mentoring programs, there remains a lack of mentorship that is equitable, individualized, and sets a clear timeline for academic milestones that will position UIM mentees at the optimal trajectory for promotion and retention. A barrier to assembling these programs is the small number of UIM among the senior faculty ranks who are able to serve in this role, given the disproportionate burden to serve on a multitude of academic committees, task forces, and workgroups to fulfill institutional mandates to diversify representation. These time-consuming services, documented in the literature as the "minority tax," are generally uncompensated and unaccounted for in terms of consideration for promotion, leadership positions, and other measures of career advancement. The Justice, Equity, Diversity, and Inclusion Academic Mentors (JAM) Council represents a novel, culturally responsive, and anti-racist approach to achieve a more equitable and inclusive institutional environment. This approach strategically leverages the intergenerational wisdom and experience of senior UIM faculty via time-protected effort with the overall goals of improving rates of promotion, retention, and career satisfaction of early career UIM colleagues. This community case study describes the rationale, resources needed, processes, and proposed workflow required to launch the JAM Council, as well as the major roles and responsibilities for JAM mentors and mentees, which may be considered by academic medical centers focused on improving diversity among the faculty ranks.

Reassessing career pathways of surgical leaders: An examination of surgical leaders' early accomplishments.

BACKGROUND: The American College of Surgeon (ACS), American Surgical Association (ASA), Association of Women Surgeons (AWS), and Society of Black Academic Surgeons (SBAS) partnered to gain insight into whether inequities found in surgical society presidents may be present earlier. METHODS: ACS, ASA, AWS, and SBAS presidents' CVs were assessed for demographics and scholastic achievements at the time of first faculty appointment. Regression analyses controlling for age were performed to determine relative differences across societies. RESULTS: 66 of the 68 presidents' CVs were received and assessed (97% response rate). 50% of AWS future presidents were hired as Instructors rather than Assistant professors, compared to 29.4% of SBAS, 25% of ASA and 29.4% of ACS. The future ACS, ASA, and SBAS presidents had more total publications than the AWS presidents, but similar numbers of 1st and Sr. author publications. CONCLUSION: Gender inequities in academic surgeon hiring practices and perceived scholastic success may be present at first hire.

Diversity in neurology leadership: Nuances and nudges.

Underrepresented in medicine (UIM) academic physicians are poorly represented among medical school faculty when compared with their proportion in the US population, receive NIH research awards less frequently, are promoted less often, indicate lower career satisfaction, and report higher social isolation, than faculty who are not under-represented. Supporting a successful and sustainable workforce of UIM academic physicians is essential in neurology, because such neurologists are more likely to engage in research to reduce disparities in neurological outcomes that affect underserved and/or low-income communities, and help improve the paucity of diverse race-ethnic participation in clinical trials. Having more diverse academic neurologists serve in such roles could bolster their careers and model possibilities for others who share similar cultures and backgrounds. Beyond leading/joining diversity affairs committees, more UIM are needed in mainstream leadership roles. In this work, we explore self-application vs. appointment/nomination opportunities and how this play a role in leadership opportunities. In addition to considering appropriate weighing of self-applications vs. appointments based opportunities, we highlight approaches and introduce the concept of nudging. Nudging, which refers to purposely increasing the visibility and appeal of particular items with the goal of boosting the odds of selecting those items, has been shown to successfully influence the process of selection, and may help level the leadership playing field for UIM in neurology.

ATG8-Interacting Motif: Evolution and Function in Selective Autophagy of Targeting Biological Processes.

Autophagy is an evolutionarily conserved vacuolar process functioning in the degradation of cellular components for reuse. In plants, autophagy is generally activated upon stress and its regulation is executed by numbers of AuTophaGy-related genes (ATGs), of which the ATG8 plays a dual role in both biogenesis of autophagosomes and recruitment of ATG8-interacting motif (AIM) anchored selective autophagy receptors (SARs). Such motif is either termed as AIM or ubiquitin-interacting motif (UIM), corresponding to the LC3-interacting region (LIR)/AIM docking site (LDS) or the UIM docking site (UDS) of ATG8, respectively. To date, dozens of AIM or UIM containing SARs have been characterized. However, the knowledge of these motifs is still obscured. In this review, we intend to summarize the current understanding of SAR proteins and discuss the conservation and diversification of the AIMs/UIMs, expectantly providing new insights into the evolution of them in various biological processes in plants.

Do I Belong Here? Confronting Imposter Syndrome at an Individual, Peer, and Institutional Level in Health Professionals.

Introduction: Imposter syndrome (IS) is a feeling of being an intellectual fraud and is common among health professionals, particularly those underrepresented in medicine. IS is accompanied by burnout, self-doubt, and beliefs of decreased success. This workshop aims to discuss the impact of IS and develop strategies to confront IS at the individual, peer, and institutional levels. Methods: During the 75-minute interactive workshop, participants listened to didactics and engaged in individual reflection, small-group case discussion, and large-group instruction. Workshop participants and facilitators included medical students, residents, fellows, faculty, staff, and program leadership. Anonymous postworkshop evaluations exploring participants' satisfaction and intentions to change their behavior were collected. Descriptive statistics were used to analyze the quantitative data, and content analysis was used to analyze participants' intentions to change their behavior. Results: The workshop was presented at three local academic conferences and accepted at one national conference. Data were collected from 92 participants. Ninety-two percent of participants felt the workshop met its objectives, and 90% felt the workshop was a valuable use of their time. Furthermore, 90% of participants stated they would apply information learned at the workshop in the future. The participants indicated an intent to change behavior on individual, peer, and institutional levels, while recognizing that barriers exist at all those levels. Discussion: This workshop proved to be an effective means to discuss strategies on how to address IS at the individual, peer, and institutional levels. The materials can be adapted for relevance to various audiences.

Multiple Functions of ATG8 Family Proteins in Plant Autophagy.

Autophagy is a major degradation process of cytoplasmic components in eukaryotes, and executes both bulk and selective degradation of targeted cargos. A set of autophagy-related (ATG) proteins participate in various stages of the autophagic process. Among ATGs, ubiquitin-like protein ATG8 plays a central role in autophagy. The ATG8 protein is conjugated to the membrane lipid phosphatidylethanolamine in a ubiquitin-like conjugation reaction that is essential for autophagosome formation. In addition, ATG8 interacts with various adaptor/receptor proteins to recruit specific cargos for degradation by selective autophagy. The ATG8-interacting proteins usually contain the ATG8-interacting motif (AIM) or the ubiquitin-interacting motif (UIM) for ATG8 binding. Unlike a single ATG8 gene in yeast, multiple ATG8 orthologs have been identified in the plant kingdom. The large diversity within the ATG8 family may explain the various functions of selective autophagy in plants. Here, we discuss and summarize the current view of the structure and function of ATG8 proteins in plants.

Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis.

Rapid, gentle isolation of 26S proteasomes from cells or tissues is an essential step for studies of the changes in proteasome activity and composition that can occur under different physiological or pathological conditions and in response to pharmacological agents. We present here three different approaches to affinity purify or to prepare proteasome-rich cell fractions. The first method uses affinity tags fused to proteasome subunits and has been useful in several cell lines for studies of proteasome structure by cryo-electron microscopy and composition by mass spectrometry. A second method uses the proteasome's affinity for a ubiquitin-like (UBL) domain and can be used to purify these particles from any cell or tissue. This method does not require expression of a tagged subunit and has proven to be very useful to investigate how proteasomal activity changes in different physiological states (e.g., fasting or aging), with neurodegenerative diseases, and with drugs or hormones that cause subunit phosphorylation. A third, simple method that is based on the 26S proteasome's high molecular weight (about 2.5 MDa) concentrates these particles greatly by differential centrifugation. This method maintains the association of proteasomes with ubiquitin (Ub) conjugates and many other loosely associated regulatory proteins and is useful to study changes in proteasome composition under different conditions.

p38-Mediated phosphorylation of Eps15 endocytic adaptor protein.

Epidermal growth factor receptor pathway substrate 15 (Eps15) has been suggested to be involved in the endocytosis of cell surface receptors, including epidermal growth factor receptor (EGFR). Eps15 is phosphorylated at Tyr-849 upon stimulation with EGF during endocytic processes. In the present study, we found that stimulation of HeLa cells with EGF or TNF-α induced transient phosphorylation of Eps15 at Ser-796. Inhibition of p38 completely blocked phosphorylation and recombinant p38α directly phosphorylated the residue. These results demonstrate a novel stress kinase-mediated signaling pathway to Eps15 endocytic adapter protein.

The Fanconi anemia ID2 complex: dueling saxes at the crossroads.

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.

Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?

The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review.

Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana.

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects.

Armadillo Repeat Containing 8alpha Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins.

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) alpha. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8alpha co-localized with HRS. ARMc8alpha promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8alpha.