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AG Cabrera

copyright: private

Proteins mediate the vast majority of biological processes, typically interacting with other proteins and/or other macromolecules rather than acting alone. Disruptions in these interactions, such as those caused by genetic mutations, can impair formation of protein complexes, leading to cellular dysfunction and physiological defects.

Despite remarkable advances in (epi)genetics, transcriptomics, and signaling research, biochemical and proteomic approaches remain essential to understanding how the cellular interactome responds and adapts, particularly under pathophysiological conditions. While significant progress has been made using these strategies, the vast amounts of generated data often lack functional validation, making interpretation in physiological contexts challenging.

Our group combines expertise in biochemistry, cell metabolism, and proteomics to address these challenges. We focus on characterizing the structure and function of disease-related mitochondrial protein complexes and advancing mass spectrometry-based approaches to identify and validate protein interactions both under normal conditions and in states of cellular stress and metabolic dysfunction.

 

Research topics of our group

Unraveling the cellular complexome in health and disease

Proteins are essential for all biological systems, often working in complexes with other proteins and macromolecules like DNA, RNA, and lipids to perform cellular functions. The collection of protein complexes, which can form either transient or stable interactions under a specific condition, is collectively referred to as complexome. Disruptions in protein complexes can impair cellular processes and contribute to various health issues. A comprehensive understanding of complexomes and protein interaction networks is essential for uncovering the molecular mechanisms underlying cell physiology and disease.

Our group studies protein interactions using Complexome Profiling (CP), a systematic, unbiased approach that combines native protein separation with tandem mass spectrometry [Cabrera-Orefice et al., 2022]. Computational clustering of the data allows us to map the composition, abundance, and organization of protein complexes in biological samples. Originally developed for mitochondrial research, CP has provided key insights into oxidative phosphorylation and the molecular basis of different mitochondrial disorders.

However, CP is not limited to mitochondria, it is a powerful tool for investigating protein-protein interactions across different cellular compartments, cell types, tissues and species. We welcome collaborations with research groups looking for a simple, flexible, and robust method to study the size, composition, and dynamics of protein complexes. This cutting-edge methodology is now available through our proteomics core facility for research groups at JLU, UKGM, and beyond.

A mesoscale model of a mitochondrion / copyright: www.cellscape.co.uk

 

Complexome Profiling: Overcoming challenges and expanding applications

Although Complexome Profiling (CP) is a powerful method, it requires significant time and resources, particularly for mass spectrometry (MS) data acquisition (~1 hour per fraction). This brings challenges when analyzing multiple experimental conditions, as each sample typically requires 32–60 fractions, leading to several days of data collection per condition.

To improve throughput and reduce MS measurement times, strategies like duplexing and triplexing with stable isotope labeling (SILAC, SILAM) [Palenikova et al., 2021] and multiplexing with isobaric labeling compounds (TMTs) [Guerrero-Castillo et al., 2021] offer promising solutions. These approaches increase efficiency and enhance proteomic quantification. SILAC is a cost-effective, widely compatible method for most cell culture conditions, while TMT labeling enables multiplexing for animal and patient samples. Our current MS setup supports both strategies, allowing seamless integration into CP studies.

Through recent collaborations, we have expanded CP applications to study DNA- and RNA-interacting protein complexes, enabling near-complete analysis of mitochondrial [Potter et al., 2023] and nuclear [Prieto-Garcia et al., 2024] complexomes within a single experiment. We are confident that further advancements in this approach will foster new and meaningful interdisciplinary collaborations, both internally and externally.

Potter, A., Cabrera-Orefice, A.*, & Spelbrink, J. N.* (2023). Nucleic acids research, 51(19), 10619–10641.

In addition to increasing throughput, automation of post-processing, visualization, and deep analysis of complexome data remains a priority for our group. While various apps and scripts exist for CP analysis, there is still a need for a user-friendly, all-in-one software solution. If you are interested in this challenge and believe you can contribute to its development, we encourage you to get in touch!

 

Understanding the biogenesis of the membrane arm of mitochondrial complex I

Mitochondrial complex I (CI) is the first and largest enzyme in the respiratory chain, catalyzing electron transfer from NADH to ubiquinone coupled to proton translocation across the inner mitochondrial membrane. In mammals, CI consists of 44 different subunits, including 14 core subunits and 30 accessory subunits with largely unclear functions. Our research mainly focuses on the assembly factors that mediate the formation of the membrane arm of CI.

Structure of mitochondrial complex I. CI consists of two main components: the peripheral arm, which extends into the mitochondrial matrix (MM) and contains the N and Q modules for NADH oxidation and ubiquinone reduction, and the membrane arm, which is embedded in the inner mitochondrial membrane (IMM) and includes the P proximal (PP) and P distal (PD) modules, responsible for proton translocation into the intermembrane space (IMS).

Up to 18 CI assembly factors have been identified across species, assisting in subunit maturation, folding, and assembly. While high-resolution structures of CI have been determined, the structures and binding sites of most assembly factors remain unknown, except for a few (e.g., fungal N7BML, NDUFAF1, CIA84, and human ACAD9/ECSIT). The presence or absence of specific assembly factors varies among species, complicating direct extrapolation of CI assembly steps.

Mutations in CI assembly factors are linked to mitochondrial diseases, including Leigh syndrome, cancer, and neurological and metabolic disorders, yet their molecular mechanisms remain unclear. Understanding their precise roles and interactions with CI intermediates is crucial for integrating the assembly pathway into a physiological context, potentially uncovering new therapeutic targets and clarifying how their dysregulation contributes to disease.

Interestingly, despite the absence of certain CI assembly factors in some taxa (e.g., plants and fungi), the overall CI structure remains highly conserved. This raises fundamental questions about species-specific proteins involved in CI assembly. In the long term, we aim to explore whether assembly factors absent in humans could serve as drug targets (e.g., for CI-containing pathogens) or be leveraged for gene therapy to enhance CI assembly in patients with mitochondrial disorders.

 

Dr. Alfredo Cabrera-Orefice / copyright: private

Group Leader and Head of the Proteomics Core Facility at the Institute of Biochemistry, JLU since June 2024. His expertise spans biochemistry, bioenergetics, proteomics and mitochondrial physiology, with a focus on mitochondrial protein assembly, molecular mechanisms, and metabolic remodelling. Alfredo has conducted research in Mexico, the Netherlands and Germany, making key contributions to mitochondrial bioenergetics and complexome profiling. His current research utilizes complexome profiling to study respiratory complex I assembly, focusing on the molecular roles, regulation, and coordination of specific assembly factors, particularly those associated with the membrane arm. He investigates the intricate role of mitochondria in mammalian and yeast species, exploring proteome and metabolic adaptations. His goal is to integrate mitochondrial function into broader cellular and physiological contexts to better understand complex biological processes and disease. Alfredo is also dedicated to advancing mass spectrometry methods for studying the cellular complexome. He actively fosters national and international collaborations, believing teamwork drives scientific progress. His research has been published in top journals such as Nature Communications, EMBO Journal, Nucleic Acids Research, Cell Metabolism, and Science. Beyond research, he is an experienced lecturer and mentor, having supervised multiple PhD and undergraduate students.

 

Selected publications

  1. Liang, C., Padavannil, A., Zhang, S., Beh, S., Robinson, D. R. L., Meisterknecht, J., Cabrera-Orefice, A., Koves, T. R., … Ho, L. (2025). Formation of I2+III2supercomplex rescues respiratory chain defects. Cell metabolism, 37(2), 441–459.e11
  2. Prieto-Garcia, C., Matkovic, V., Mosler, T., Li, C., Liang, J., Oo, J. A., Haidle, F., Mačinković, I., Cabrera-Orefice, A., Berkane, R., … Dikic, I. (2024). Pathogenic proteotoxicity of cryptic splicing is alleviated by ubiquitination and ER-phagy. Science,386(6723), 768–776.
  3. Heidler, J.*, Cabrera-Orefice, A.*, Wittig, I.*, Heyne, E., Tomczak, J. N., … Szibor, M. (2024). Hyperbaric oxygen treatment reveals spatiotemporal OXPHOS plasticity in the porcine heart. PNAS nexus, 3(6), pgae210. *Equal contribution.
  4. Castañeda-Tamez, P., Chiquete-Félix, N., Uribe-Carvajal, S.*, & Cabrera-Orefice, A.* (2024). The mitochondrial respiratory chain from Rhodotorula mucilaginosa, an extremophile yeast. Biochim Biophys Acta Bioenergetics, 1865(2), 149035. *Shared corresponding authorship.
  5. Potter, A., Cabrera-Orefice, A.*, & Spelbrink, J. N.* (2023). Let's make it clear: systematic exploration of mitochondrial DNA- and RNA-protein complexes by complexome profiling. Nucleic acids research, gkad697. *Shared corresponding authorship.
  6. Salscheider, S. L.*, Gerlich, S.*, Cabrera-Orefice, A.*, Peker, E., … Riemer, J. (2022). AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5. The EMBO journal, e110784. *Equal contribution
  7. Cabrera-Orefice, A.*, Potter, A., Evers, F., Hevler, J. F., Guerrero-Castillo, S*. (2022). Complexome Profiling-Exploring Mitochondrial Protein Complexes in Health and Disease.  Cell Dev. Biol.9:796128. *Shared corresponding authorship
  8. Evers, F., Cabrera-Orefice, A., Elurbe, D. M., Kea-Te Lindert, M., Boltryk, S. D., Voss, T. S., Huynen, M. A., Brandt, U., Kooij, T. (2021) Composition and stage dynamics of mitochondrial complexes in falciparum. Nature Communications 12, 3820.
  9. Cabrera-Orefice A., Yoga E. G., Wirth C., Siegmund K., Zwicker K., Guerrero-Castillo S., Zickermann V., Hunte C., Brandt U. (2018) Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps. Nature communications 9(1): 4500.
  10. Cabrera-Orefice A, Chiquete-Félix N, Espinasa-Jaramillo J, Rosas-Lemus M, Guerrero-Castillo S, Peña A, Uribe-Carvajal S. (2014) The branched mitochondrial respiratory chain from Debaryomyces hansenii: Components and supramolecular organization. Biochim Biophys Acta Bioenergetics 1837(1):73-84.

For a complete list of publications of Alfredo Cabrera-Orefice, see Pubmed.

 

Team members

  • Biol. Uwe Schubert – Lab Manager
  • Tanvika Katyayan, B.Sc. – Student Assistant

 

Are you interested in joining our group? 

Send an e-mail with your CV to Dr. Cabrera-Orefice.

 

Medical students: please inquire for vacancies for MD thesis!

Diploma/Master students are most welcome to join our team!

We are at all times interested in applications of potential PhD students and/or Postdocs who are willing to join a productive and competitive team with a friendly atmosphere.

 

Contact

alfredo.cabrera