First person – Nikita Sergejevs

Nikita Sergejevs

How would you explain the main findings of your paper in lay terms?

This project addresses an important question in cell biology: how are protein quality control substrates selected for degradation? Specifically, we explore the role of intramembrane proteases, which are implicated in this process by ill-defined mechanisms. We focus on the signal peptide peptidase (SPP), previously identified by our laboratory as being required for the ER-associated degradation (ERAD) of a mutant version of human lanosterol demethylase, CYP51A1TM. We find that both CYP51A1TM and endogenous CYP51A1 are SPP substrates and that SPP-dependent trimming of the N-terminus of CYP51A1 is a trigger for subsequent clearance of these substrates by ERAD. We demonstrate that SPP binds directly to these substrates by recognising an amphipathic helix at their N-terminal region. Curiously, SPP only recognises CYP51A1, a type II membrane protein, when its amphipathic helix is aberrantly inserted in the type II configuration. This agrees with SPP substrate preferences in terms of membrane protein topology and explains why only a fraction of the endogenous CYP51A1 is degraded whereas all mutant CYP51A1TM is eliminated. These findings reveal a new role for SPP in monitoring protein topology and triggering the degradation of substrates that adopt incorrect or potentially non-functional conformations. This insight offers a significant advancement in our understanding of how intramembrane proteases contribute to membrane protein quality control.

Were there any specific challenges associated with this project? If so, how did you overcome them?

One of the challenges that we encountered during the project was the assessment of the metastable nature of the amphipathic helix of CYP51A1. The common assay to monitor dual topology of the amphipathic helix would have been to use an epitope tag at the N-terminus of the target protein. However, all our attempts to introduce an N-terminal tag resulted in substrate stabilization. Over the course of the project, I tried using numerous epitope tags with and without flexible linkers, but any modification of the N-terminal part of the substrate abolished its dependency on not only SPP but also on the RNF185–MBRL ERAD complex for degradation. We suspected that the addition of an N-terminal tag would destroy the metastable nature of the amphipathic helix and ‘lock’ it in the ER lumen, where it cannot be recognized by SPP and be engaged in ERAD.

Because the addition of the N-terminal tag was not an option, we resorted to using a glycan acceptor sequence and used pulse-chase labelling to capture the event of amphipathic helix recognition by SPP. This strategy offered the advantage of allowing the conditional block of glycosylation using tunicamycin, a potent inhibitor of N-linked glycosylation. In addition, we supported our hypothesis with AI-guided structural predictions, which showed that the amphipathic helix did not interact with the rest of the protein and adopted multiple conformations.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

Early in the project, we decided to test whether SPP had a general role in regulating the stability of cytochrome P450 (CYP450) enzymes localized to the ER by developing a library of simplified model substrates consisting of the N-terminal membrane domains of 20 CYP450s. We were successful at identifying substrates of various E3 ligase complexes, including RNF185–MBRL, MARCHF6 and HRD1. Remarkably, although these substrates were seemingly similar in their domain organization and topology, only one substrate required SPP activity for its degradation – CYP51A1TM. Further analysis of the various CYP450s revealed an N-terminal amphipathic helix present within CYP51A1 that was not apparent in other CYP450 family members. This allowed us to proceed with identifying the metastable behaviour of this amphipathic helix and dissect the mechanism by which CYP51A1 is recognised by SPP for quality control.

Why did you choose Journal of Cell Science for your paper?

Journal of Cell Science has a high-quality peer review process that was quick and fair; the editorial process was supportive and enhanced the academic value of our publication. The process of manuscript submission is extremely well organised, and the standard of published work is high.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

I was incredibly fortunate to have dedicated colleagues and mentors around me throughout my DPhil journey − their collective passion for science and drive for success made the Carvalho laboratory and the Sir William Dunn School of Pathology exceptional places to work and develop as a scientist. The environment at the Sir William Dunn School of Pathology is truly inspirational – here, I really felt the true power of academic research − highlighting the importance of collaboration, innovation and great attention to detail.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

While still in secondary school in Latvia, I was a proactive student and managed to secure an internship at the Department of Material Science and Applied Chemistry under the supervision of Professor Erika Bizdena. This early experience sparked my interest in academic research. My work led to an invitation to represent Latvia at the European Union Contest for Young Scientists (EUCYS), where I presented my research project and exchanged ideas with young scientists from across the globe, solidifying my love of interdisciplinary research. I then moved to the UK to pursue a degree in Biochemistry at the University of Warwick. During my undergraduate studies, attending lectures on protein targeting by Professor Lorenzo Frigerio stimulated my passion for mechanistic biochemistry. This, together with excellent academic achievements, led me to receive the Amgen Scholarship to conduct research in the laboratory of Professors F.-Ulrich Hartl and Manajit Hayer-Hartl in Martinsried, where I honed my laboratory techniques and acquired new skills, such as protein expression and purification. These immersive experiences − being a member of a team driving cutting-edge research, engaging in academic discussions and benefitting from excellent mentorship − solidified my passion for scientific inquiry and ultimately guided me towards an advanced research degree. My progression to DPhil studies in the laboratory of Professor Pedro Carvalho felt like a natural next step, and I am thoroughly enjoying this experience.

Who are your role models in science? Why?

As a member of the Sir William Dunn School of Pathology, I am honoured to walk the same halls once frequented by the pioneering scientists behind the discovery of penicillin, including Professors Howard Florey and Ernst Chain. I would also like to highlight an often-overlooked hero of this story, Norman Heatley, whose extraordinary contributions to this monumental discovery are often underappreciated. His remarkable work ethic, meticulous note-taking and relentless drive for success, often from behind the scenes, make Norman Heatley an inspiring role model in science.

What's next for you?

I am currently in the final stages of preparing for my thesis defence and completing the second project of my DPhil, which focuses on membrane protein recognition for quality control. Throughout my academic journey, I have developed a strong interest in translating fundamental scientific discoveries into clinical applications. This interest, combined with my extensive volunteering experience in healthcare settings, has inspired me to consider returning to the lecture hall to pursue a medical degree. I am excited about the possibility of combining my research background with clinical practice to make a more direct impact on patient care.

Tell us something interesting about yourself that wouldn't be on your CV

Outside of the laboratory setting, I enjoy playing sports; in my second year of DPhil studies, I achieved the title of British Universities Judo Champion.