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The electron transport chain (ETC) is used by cells to create an electrochemical proton gradient which can be used by the ATP synthase to produce ATP. ETC, also called respiratory chain, is formed in mitochondria by four complexes (complex I-IV) and mediated by two electron carriers: cytochrome c and ubiquinone. Electrons are passed from one complex to another in a series of redox reactions coupling proton pumping from the negative (N) side of the membrane to the positive (P) side. Complex I can introduce electrons into the ETC by oxidizing NADH to NAD+ and reducing quinone (Q) to quinol (QH2). The process accomplishes pumping of four protons across the membrane. Complex II is another electrons entry point. It catalyzes the oxidation of succinate to fumarate while reducing Q to QH2. Complex III, also called cytochrome bc1 complex, can transfer the electrons from QH2 to cytochrome c and couple to proton pumping. In complex III the Q-cycle contributes four proton translocations: two protons are required for the reduction of one quinone to a quinol and two protons are released to the P side. Complex IV (cytochrome c oxidase), the terminal complex of the ETC, catalyzes the electron transfer to oxygen and pumps four protons to the P side. Structures of ETC complexes are available. However, the structure of a hyperthermophilic cytochrome bc1 complex has not been elucidated till now. Additionally, the dimeric crystal structure of cytochrome c oxidase from bovine has been discussed controversially.
To build up a functional complex, cofactors are required. The active site of A- and B-type cytochrome c oxidases contain the high spin heme a which is synthesized by the integral membrane protein heme A synthase (HAS). HAS can form homooligomeric complexes and its oligomerization is essential for the biological function of HAS. HAS is evolutionarily conserved among prokaryotes and eukaryotes. Despite its importance, little is known about the detailed structural properties of HAS oligomers.
During my PhD studies, I focused on the cytochrome c oxidase (AaCcO), the cytochrome bc1 complex (Aabc1) and the heme A synthase (AaHAS) from Aquifex aeolicus. This organism is one of the most hyperthermophilic ones and can live at extremely high temperatures, even up to 95 °C. Respiratory chain complexes provide energy for the metabolism of organisms, and their structures have been studied extensively in the past few years. However, there has been a lack of atomic structures of complexes from hyperthermophilic and ancient bacteria, so little is known about the mechanism of these macromolecular machines under hyperthermophilic conditions. Therefore, my PhD studies had four main objectives: 1) to structurally and functionally characterize AaCcO, 2) to reveal the mechanism of Aabc1 thermal stability based on its structure, 3) to determine the oligomerization of AaHAS, 4) to provide valuable insights into the relationship between function and oligomerization of AaHAS.
1) Structure of AaCcO
Heme-copper oxidases (HCOs) catalyze the oxygen reduction reaction being the terminal enzymes in the plasma membranes in many prokaryotes or of the aerobic respiratory chain in the inner mitochondrial membrane. By coupling this exothermic reaction to proton pumping across the membrane to the P side, they contribute to the establishment of an electrochemical proton gradient. The energy in the proton electrochemical proton gradient is used by the ATP synthase to generate ATP. HCOs are classified into three major families: A, B and C, based on phylogenetic comparisons. The well-studied aa3-type cytochrome c oxidase from Paracoccus denitrificans (P. denitrificans) represents A-family HCOs. So far, the only available structure of the ba3-type cytochrome c oxidase from Thermus thermophilus represents the B-family of HCOs. This family contains a number of bacterial and archaeal oxidases. The C-family contains only cbb3-type cytochrome c oxidases.
The AaCcO is one of the ba3-type cytochrome c oxidases. Based on the genomic DNA sequence analysis, it has been revealed that A. aeolicus possesses two operons coding for cytochrome c oxidases (two different subunit I genes, two different subunit II genes and one subunit III gene). So far, only subunits CoxB2 and CoxA2 were identified. The presence of the additional subunit IIa was reported in 2012. Moreover, a previous paper reported that AaCcO can use horse heart cytochrome c and decylubiquinol as electron donors and the typical cytochrome c oxidase inhibitor cyanide does not block the reaction completely.
In the course of my PhD studies, I performed heterologous expression of AaCcO in Pseudomonas stutzeri (P. stutzeri) and co-expression with AsHAS in Escherichia coli, respectively. The subcomplex CoxA2 and CoxB2 can be purified from P. stutzeri, however, it lacks heme A. Additionally, a protocol for the heterologous production of cytochrome c555 from A. aeolicus was established. In parallel, I also purified the AaCcO from native membranes according to previously reported methods with some modifications. The activity of AaCcO with its native substrate, cytochrome c555, was 14 times higher than with horse heart cytochrome c.
To enable a detailed investigation and comparison of AaCcO and other cytochrome c oxidases, the cryo-EM structure of AaCcO was determined to 3.4 Å resolution. It shows that the three subunits CoxA2, CoxB2, and IIa are tightly bound together to form a dimer in the membrane. Surprisingly, CoxA2 contains two additional TMHs (TMH13 and TMH14) to enhance the protein stability. The cofactors heme a3, heme b, CuA and CuB are also identified. Interestingly, two molecules of 1,4-naphthoquinone and cardiolipin were observed in the dimer interface. Based on the structure analysis, the AaCcO possesses only the K-pathway for proton delivery to the active site and proton pumping.
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Autophagy is an important degradation pathway mediating the engulfment of cellular material (cargo) into autophagosomes followed by degradation in autophagosomes.
Different stress stimuli, e.g. nutrient deprivation, oxidative stress or organelle damage, engage autophagy to maintain cellular homeostasis, recycle nutrients or remove damaged cell organelles. Autophagy not only degrades bulk cytoplasmic material but also selective autophagic cargo, for example lysosomes (lysophagy), mitochondria (mitophagy), ER (ER-phagy), lipid droplets (lipophagy), protein aggregates (aggrephagy) or pathogens (xenophagy). Selective autophagy pathways are regulated by selective autophagy receptors which bind to ubiquitinated cargo proteins and link them to LC3 on the autophagosomal membrane.
Ubiquitination is an essential post-translational modification controlling different cellular processes such as proteasomal and lysosomal degradation or innate immune signaling.
M1-linked (linear) poly-Ubiquitin (poly-Ub) chains are exclusively assembled by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the M1 poly-Ub-specific OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN). In addition to key functions in innate immune signaling and nuclear factor-κB (NF-κB) activation, M1 ubiquitination is also implicated in the regulation of autophagy.
LUBAC and OTULIN control autophagy initiation and maturation and the autophagic clearance of invading bacteria via xenophagy. However, additional functions of LUBAC- and OTULIN-regulated M1 ubiquitination in autophagy are largely unknown and it also remains unexplored if LUBAC and OTULIN control other selective autophagy pathways in addition to xenophagy. This study aimed to unravel the role of LUBAC- and OTULIN-controlled M1 ubiquitination in bulk and selective autophagy in more detail.
In this study, characterization of OTULIN-depleted MZ-54 glioblastoma (GBM) cells revealed that OTULIN deficiency results in enhanced LC3 lipidation in response to autophagy induction and upon blockade of late stage autophagy with Bafilomycin A1 (BafA1). Furthermore, electron microscopy analysis showed that OTULIN-deficient cells have an increased number of degradative compartments (DGCs), confirming enhanced autophagy activity upon loss of OTULIN. APEX2-based autophagosome content profiling identified various OTULIN-dependent autophagy cargo proteins. Among these were the autophagy receptor TAX1BP1 which regulates different forms of selective autophagy (e.g. lysophagy, aggrephagy) and the glycan-binding protein galectin-3 which serves key functions in lysophagy, suggesting a role of OTULIN and M1 poly-Ub in the regulation of aggrephagy and lysophagy.
Abstract 2
To study aggrephagy, protein aggregation was induced with puromycin which causes premature termination of translation and accumulation of defective ribosomal products (DRiPs). Loss of OTULIN increased the number of M1 poly-Ub-positive foci and insoluble proteins and reduced the levels of soluble TAX1BP1 and p62 in response to puromycin-induced proteotoxic stress.
Intriguingly, upon induction of lysosomal membrane permeabilization (LMP) with the lysosomotropic drug L-Leucyl-L-Leucine methyl ester (LLOMe), M1 poly-Ub strongly accumulated at damaged lysosomes and colocalized with TAX1BP1- and galectin-3-positive puncta. M1 poly-Ub-modified lysosomes formed a platform for NF-κB essential modulator (NEMO) and inhibitor of κB (IκB) kinase (IKK) complex recruitment and local NF-κB activation in a K63 poly-Ub- and OTULIN-dependent manner. Furthermore, inhibition of lysosomal degradation enhanced LLOMe-induced cell death, suggesting pro-survival functions of lysophagy following LMP. Enrichment of M1 poly-Ub at damaged lysosomes was also observed in human dopaminergic neurons and in primary mouse embryonic cortical neurons, confirming the importance of M1 poly-Ub in the response to lysosomal damage.
Together, these results identify OTULIN as a negative regulator of autophagy induction and the autophagic flux and reveal OTULIN-dependent autophagy cargo proteins.
Furthermore, this study uncovers novel and important roles of M1 poly-Ub in the response to lysosomal damage and local NF-κB activation at damaged lysosomes.
Die Bande q23 auf Chromosom 11 ist in zahlreiche reziproke chromosomale Translokationen verwickelt. Diese sind dominant mit dem Krankheitsphänotyp einer AML, ALL und seltener mit malignen Lymphomen und myelodysplastischen Syndromen assoziiert. Mittlerweile sind fünfundachtzig cytogenetische Aberrationen der Bande 11q23 bekannt. Das auf 11q23 betroffene Gen wird als das Mixed Lineage Leukemia (MLL), Acute Lymphoblastic Leukemia (ALL-1), Human Homolog of trithorax (HRX) oder als Human Trithorax 1 (Htrx1) bezeichnet. Die häufigsten Partnergene des MLL sind AF4 (40 %), AF9 (27 %), sowie ENL, AF6, ELL und AF10 (4-7 %).
Die Bruchpunkte von t(11;V) Translokationen sind nicht gleichmäßig über das gesamte, 92 kb große humane MLL-Gen verteilt, sondern liegen alle in der 8,3 kb großen Bruchpunktsregion Bpr. Auch innerhalb der Bpr ist die Verteilung der Translokationsbruchpunkte nicht homogen. Die Bruchpunkte von Patienten mit de novo Leukämien und einem Alter über einem Jahr liegen mehrheitlich in der 5’-Hälfte der Bpr, dem Subcluster I. Dagegen liegen die Bruchpunkte von Patienten mit therapiebedingten Leukämien und einem Alter unter einem Jahr überwiegend in der 3’-Hälfte der Bpr, dem Subcluster II.
Neuere Forschungsergebnisse zeigten, daß DNA-Doppelstrangbrüche auf zwei verschiedenen Chromosomen eine hinreichende Voraussetzung für das Entstehen chromosomaler Translokationen sind. Aufgrund der inhomogenen Verteilung der Translokationsbruchpunkte im MLL-Gen stellte sich die Frage, ob bestimmte Regionen dieses Gens für DNA-Doppelstrangbrüche prädisponiert sind. Interessanterweise ist Subcluster II extrem sensitiv gegenüber DNA-Doppelstrangbrüchen, die durch cytotoxische Agenzien oder Apoptose-auslösende Ereignisse induziert werden können. In unserer Arbeitsgruppe konnte eine etwa 200 bp große Region lokalisiert werden, über die sich nahezu alle Etoposid-induzierten DNA-Doppelstrangbrüche verteilten.
In dieser Arbeit konnte gezeigt werden, daß die Bildung von DNA-Doppelstrangbrüchen in dieser Region durch die Gabe eines Caspase-Inhibitors gehemmt werden kann. Eine Etoposid-induzierte Protein-DNA-Wechselwirkung konnte allerdings nicht nachgewiesen werden. In der Literatur fanden sich Hinweise darauf, daß Subcluster II im Gegensatz zu Subcluster I eine verstärkte Histonacetylierung aufweist. Basierend auf diesen Hinweisen sollte die Arbeitshypothese untersucht werden, ob Subcluster II einen geninternen Promotor des MLL-Gens darstellt.
Die potentielle Promotorregion wurde zunächst durch Computeranalysen eingegrenzt. Mit RT-PCR Experimenten wurde anschließend der potentielle geninterne Promotor des murinen Mll-Gens in einer murinen Fibroblastenzellinie lokalisiert, die einen Transkriptionsstop und eine Polyadenylierungssequenz in Exon 4 des Mll-Gens trug. Um die am Mausmodell gewonnenen Erkenntnisse auch im humanen System zu überprüfen, wurde die geninterne Promotorregion des humanen MLL Gens vor ein Luciferasereportergen kloniert. Durch RTPCR konnte der geninterne Transkriptionsstart im Subcluster II des humanen MLL-Gens lokalisiert werden. Damit konnte zum ersten Mal gezeigt werden, daß Transkriptionsinitiation und genetische Instabilität im Subcluster II des humanen MLL-Gens kolokalisieren.
Durch Deletionsmutanten wurde die Bedeutung der einzelnen Module dieser Promotorregion ermittelt. Dabei zeigte sich, daß die Anwesenheit von zwei retromobilen Elementen eine Enhancer-Funktion haben. Demgegenüber zeigte die homologe murine Sequenz, die in unserer Arbeitsgruppe gleichzeitig von S. Scharf untersucht wurde und für die keine erhöhte Anfälligkeit für DNA-Doppelstrangbrüche bekannt ist, nur eine schwache Promotoraktivität. Dies weist auf einen Zusammenhang zwischen der genetischen Instabilität von Subcluster II und der Rate geninterner Transkriptionsinitiationsprozesse hin.
Das Protein, für das das Transkript des geninternen murinen Promotors kodiert, wurde mittels immunhistologischer und Western Blot Experimente nachgewiesen. Dabei konnte gezeigt werden, daß dieses Protein, wie auch das MLL-Protein, proteolytisch durch Taspase1 und daß sich ein Mini-MLL-Komplex bildet.
The formation of amyloid-β oligomers plays a key role in the onset of Alzheimer’s disease. We investigated the aggregation of amyloid-β oligomers by mass spectrometry and ion mobility spectrometry, revealing those structural properties, which lead to the formation of mature fibrils. We can show that the arrangement of the first oligomers is crucial for the topology of the resulting species, leading to the formation of non-toxic aggregates or fibrils.
B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
The members of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) transporter superfamily mediate export of a wealth of molecules of physiological and pharmacological importance. According to the Transporter Classification Database (TCDB), the MOP superfamily is mainly categorized into six distantly related families functionally characterized families: the multidrug and toxic compound extrusion (MATE), the polysaccharide transporter (PST), the oligosaccharidyl-lipid flippase (OLF), the mouse virulence factor (MVF) the agrocin 84 antibiotic exporter (AgnG), and the progressive ankylosis (Ank) family. Among these, the multidrug resistance MATE family transporters are most ubiquitous, being present in all domains of life: Archaea, Bacteria and Eukarya. As secondary active transporters, they utilize transmembrane electrochemical ion gradients of Na+ and/or H+ in order to drive the efflux of xenobiotics or cytotoxic metabolic waste products with specificity mainly for polyaromatic and cationic substrates. Active efflux of drugs and toxic compounds carried out by multidrug transporters is one of the strategies developed by bacterial pathogens to confer multidrug resistance. MATE proteins provide resistance to, e.g., fluoroquinolone, aminoglycoside antibiotics, and anticancer chemotherapeutical agents, thus serving as promising pharmacological targets for tackling a severe global health issue. Based on their amino acid sequence similarity, the MATE family members are classified into the NorM, the DNA-damage-inducible protein F (DinF), and the eukaryotic subfamilies. Structural information on the alternate conformational states and knowledge of the detailed mechanism of the MATE transport are of great importance for the structure-aided drug design. Over the past decade, the crystal structures of representative members of the NorM, DinF and eukaryotic subfamilies have been presented. They all share similar overall architecture comprising 12 transmembrane helices (TMs) divided into two domains, the N-terminal domain (TMs 1-6) and the C-terminal domain (TMs 7-12), connected by a cytoplasmic loop between TM6 and TM7 (Fig. II.1). Since all available MATE family structures are known only in V-shaped outward-facing states with the central binding cavity open towards the extracellular side, a detailed understanding of the complete transport cycle has remained elusive. In order to elucidate the underlying steps of the MATE transport mechanism, structures of distinct intermediates, particularly inward-facing conformation, are required.In my PhD project, structural and functional studies have been performed on a MATE family (DinF subfamily) transporter, PfMATE, from the hyperthermophilic and anaerobic archaeon Pyrococcus furiosus. This protein was produced homologously in Pyrococcus furiosus as well as heterologously in Escherichia coli, and used for the subsequent purification and crystallization trials by the vapor diffusion (VD) and lipidic cubic phase (LCP) method. To the best of my knowledge, PfMATE is the first example of a successful homologous production of a membrane protein in P. furiosus. Due to the very low final amount of the purified protein from the native source, the heterologously produced PfMATE samples were typically used for the extensive structural studies. Crystal structures of PfMATE have been previously determined in an outward-facing conformation in two distinct states (bent and straight) defined on the arrangement of TM1. A pH dependent conformational transition of this helix regulated by the protonation state of the conserved aspartate residue Asp41 was proposed. However, it has been discussed controversially, leading to the hypothesis about TM1 bending to be rather affected by interactions with exogenous lipids (monoolein) present under the crystallization conditions. Based on these open questions, an experimental approach to investigate the role of lipids as structural and functional modulators of PfMATE has been taken in the course of my PhD project. The interplay between membrane proteins and lipids can affect membrane protein topology, structure and function. Considering differences between archaeal and bacterial lipid composition, cultivation of P. furiosus cells and extraction of its lipids was followed by the mass spectrometry (MS) based lipidomics for identification of individual lipid species in the archaeal extract. In order to assess the effects of lipids on PfMATE, different lipid molecules were used for co-purification and co-crystallization trials. This dissertation presents a workflow leading to the structure determination of a MATE transporter in the long sought-after inward-facing state, which has been achieved upon purification and crystallization of the heterologously produced PfMATE in the presence of lipids from its native source P. furiosus. Also, the PfMATE outward-facing state obtained from the crystals grown at the acidic pH conditions sheds light on the previously proposed pH-dependent structural alterations within TM1. It is interesting to note that the inward and outward-facing states of PfMATE were obtained from the crystals grown under similar conditions, but in the presence and absence of native lipids, respectively. This observation supports the hypothesis about physiologically relevant lipids to act as conformational modulators or/and a new class of substrates, expanding the substrate spectrum of the MATE family transporters. Comparative analysis of two PfMATE states reveals that transition from the outward to the inward-facing state involves rigid body movements of TMs 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TMs 1 and 7 to their respective bundles and their conformational flexibility. Local fluctuations within TM1 in the inward-facing structure, including bending and unwinding in the intracellular half of the helix, invoke its highly flexible nature, which is suitable for ion and substrate gating.
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Druggability Evaluation of the Neuron Derived Orphan Receptor (NOR-1) Reveals Inverse NOR-1 Agonists
(2022)
The neuron derived orphan receptor (NOR-1, NR4A3) is among the least studied nuclear receptors. Its physiological role and therapeutic potential remain widely elusive which is in part due to the lack of chemical tools that can directly modulate NOR-1 activity. To probe the possibility of pharmacological NOR-1 modulation, we have tested a drug fragment library for NOR-1 activation and repression. Despite low hit-rate (<1 %), we have obtained three NOR-1 ligand chemotypes one of which could be rapidly expanded to an analogue comprising low micromolar inverse NOR-1 agonist potency and altering NOR-1 regulated gene expression in a cellular setting. It confirms druggability of the transcription factor and may serve as an early tool to assess the role and potential of NOR-1.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
An automated NMR chemical shift assignment algorithm was developed using multi-objective optimization techniques. The problem is modeled as a combinatorial optimization problem and its objective parameters are defined separately in different score functions. Some of the heuristic approaches of evolutionary optimization are employed in this problem model. Both, a conventional genetic algorithm and multi-objective methods, i.e., the non-dominated sorting genetic algorithms II and III (NSGA2 and NSGA3), are applied to the problem. The multi-objective approaches consider each objective parameter separately, whereas the genetic algorithm followed a conventional way, where all objectives are combined in one score function. Several improvement steps and repetitions on these algorithms are performed and their combinations are also created as a hyper-heuristic approach to the problem. Additionally, a hill-climbing algorithm is also applied after the evolutionary algorithm steps. The algorithms are tested on several different datasets with a set of 11 commonly used spectra. The test results showed that our algorithm could assign both sidechain and backbone atoms fully automatically without any manual interactions. Our approaches could provide around a 65% success rate and could assign some of the atoms that could not be assigned by other methods.