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The scope of this work is to develop a novel single-molecule imaging technique by combining atomic force microscopy (AFM) and optical fluorescence microscopy. The technique is used for characterizing the structural properties of multi-protein complexes. The high-resolution fluorescence microscopy and AFM are combined (FIONA-AFM) to allow for the identification of individual proteins in such complexes. This is achieved by labeling single proteins with fluorescent dyes and determining the positions of these fluorophores with high precision in an optical image. The same area of the sample is subsequently scanned by AFM. Finally, the two images are aligned and the positions of the fluorophores are displayed on top of the topographical data. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, fluorescence and AFM information can be aligned with an accuracy better than 10 nm, which is sufficient to identify single fluorescently labeled proteins in most multi-protein complexes. The limitations of localization precision and accuracy in fluorescence and AFM images are investigated, including their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two complementary techniques opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5–10 nm) information about the conformational properties of multi-protein complexes while the fluorescence can indicate spatial relationships of the proteins within the complexes. Additionally, computer simulations are performed in order to validate the accuracy of the registration algorithm.
Gegenstand der vorliegenden Untersuchungen waren das retinaspezifische palmitoylierte Membranprotein 4 (engl. membrane palmitoylated protein 4, MPP4) und das ubiquitär exprimierte MPP5, die beide zur großen Familie der Membran-assoziierten Guanylatkinasen (engl. membrane-associated guanylate kinases, MAGUKs) gehören. Beide Proteine haben wichtige organisatorische Funktionen als Adapterproteine in der Netzhaut. MPP4 ist am Aufbau von präsynaptischen Proteinkomplexen in den Photorezeptor-Ribbonsynapsen beteiligt. Ein Fehlen von MPP4 in Mäusen führt zum Verlust von einigen präsynaptischen Proteinen von der Ribbonsynapse, was eine wichtige Rolle von MPP4 für die Organisation dieses Komplexes indiziert. Um neue Komponenten dieses Multiproteinkomplexes zu identifizieren, wurde in der vorliegenden Arbeit ein proteomischer Ansatz etabliert. Dazu wurde der MPP4-assoziierte Proteinkomplex aus Proteinextrakten der bovinen Retina durch Immunaffinitätschromatographie isoliert, mittels zweidimensionaler Gelelektrophorese aufgetrennt und resultierende Protein-spots massenspektrometrisch analysiert. Diese Untersuchungen identifizierten 18 kopräzipitierte Proteine. Darunter waren der bekannte Interaktionspartner Veli3 und das MAGUK-Protein PSD95. Für letzteres konnte gezeigt werden, dass speziell die β-Isoform von PSD95 mit MPP4 über die N-terminalen L27-Domänen heterodimerisiert. Daneben konnte die Assoziation von MPP4 mit Hsc70 und Recoverin durch unabhängige Bindungs-assays verifiziert werden, wobei diese Interaktionen indirekt waren und möglicherweise von retinaspezifischen Proteinen vermittelt werden. Diese Ergebnisse unterstützen die herausragende Rolle von MPP4 als Gerüstprotein für die Organisation eines Proteinkomplexes der Photorezeptorsynapse, der an der Regulierung der Ca2+-Homöostase in den präsynaptischen Enden, sowie vermutlich auch an der Exozytose der synaptischen Vesikel beteiligt ist. Die übrigen kopräzipitierten Proteine waren Bestandteile des Zytoskeletts und verschiedener Signalwege und wahrscheinlich unspezifische Kontaminanten. Das MPP5-Protein ist Teil eines evolutionär konservierten Proteinkomplexes, der für die Entstehung und Aufrechterhaltung der apikobasalen Polarität in Epithelzellen eine wichtige Rolle spielt und in der Netzhaut für die speziellen Zellverbindungen zwischen Müller-Gliazellen und Photorezeptoren in der externen limitierenden Membran essentiell ist. Mutationen, die zu Störungen in der Funktion dieses Proteinkomplexes führen, verursachen retinale Defekte bei Mensch (z.B. Retinitis Pigmentosa, Lebersche kongenitale Amaurose), Maus, Zebrafisch und Fruchtfliege. Die PDZ-Domäne von MPP5 bindet an den C-Terminus der CRB-Proteine. In der vorliegenden Arbeit wurde die Auswirkung einer Punktmutation in der PDZ-Domäne von MPP5 (V301N), funktionell untersucht. Diese hat im Zebrafisch eine fehlende Photorezeptoradhäsion und die Zerstörung der retinalen Architektur zur Folge. Es wurde gezeigt, dass die V301N-Mutation die Interaktion zwischen MPP5 und den drei CRB-Isoformen verhindert. Außerdem führt sie zu einer Fehllokalisierung von heterolog exprimiertem MPP5V301N in MDCK-Zellen, die zudem einen erhöhten transepithelialen Widerstand aufweisen. Über quantitative real-time PCR wurde in diesen Zellen eine verminderte Genexpression von Untereinheiten des apikalen epithelialen Natriumkanals ENaC und der basolateralen Na+/K+-ATPase festgestellt, was die Ursache für einen verminderten Ionenfluss durch die Zellschicht darstellen könnte. Somit übt heterolog exprimiertes MPP5V301N in MDCK-Zellen einen dominanten Effekt aus, der möglicherweise über einen Einfluss auf die Genregulation bestimmter Proteine vermittelt wird. Zur Untersuchung welche Auswirkungen die MPP5V301N-Mutation in vivo hat, wurde eine MPP5V301N-knock-in-Mauslinie generiert. Die bisherige Charakterisierung der Mäuse zeigte bei heterozygoten Tieren keinen äußerlich erkennbaren Phänotyp, wohingegen homozygote Mäuse nicht lebensfähig waren. Vermutlich führt das Vorkommen beider mutanter Allele in frühen embryonalen Stadien zum Abstoßen der Embryos. Das Mausmodell bietet die Basis für weitere Untersuchungen über die molekularen Konsequenzen einer Expression von MPP5V301N in der Netzhaut und anderen Geweben und wird Beiträge zur Aufklärung von Entstehungsmechanismen CRB1-MPP5-assoziierter Netzhauterkrankungen liefern.
Complex formation between macromolecules constitutes the foundation of most cellular processes. Most known complexes are made up of two or more proteins interacting in order to build a functional entity and therefore enabling activities which
the single proteins could otherwise not fulfill. With the increasing knowledge about
noncoding RNAs (ncRNAs) it has become evident that, similar to proteins, many of
them also need to form a complex to be functional. This functionalization is usually executed by specific or global RNA-binding proteins (RBPs) that are specialized
binders of a certain class of ncRNAs. For instance, the enterobacterial global RBPs
Hfq and ProQ together bind >80 % of the known small regulatory RNAs (sRNAs),
a class of ncRNAs involved in post-transcriptional regulation of gene expression.
However, identification of RNA-protein interactions so far was performed individually by employing low-throughput biochemical methods and thereby hindered the discovery of such interactions, especially in less studied organisms such
as Gram-positive bacteria. Using gradient profiling by sequencing (Grad-seq), the
present thesis aimed to establish high-throughput, global RNA/protein complexome resources for Escherichia coli and Streptococcus pneumoniae in order to provide a
new way to investigate RNA-protein as well as protein-protein interactions in these
two important model organisms.
In E. coli, Grad-seq revealed the sedimentation profiles of 4,095 (∼85 % of
total) transcripts and 2,145 (∼49 % of total) proteins and with that reproduced
its major ribonucleoprotein particles. Detailed analysis of the in-gradient distribution of the RNA and protein content uncovered two functionally unknown
molecules—the ncRNA RyeG and the small protein YggL—to be ribosomeassociated. Characterization of RyeG revealed it to encode for a 48 aa long, toxic protein that drastically increases lag times when overexpressed. YggL was shown to
be bound by the 50S subunit of the 70S ribosome, possibly indicating involvement
of YggL in ribosome biogenesis or translation of specific mRNAs.
S. pneumoniae Grad-seq detected 2,240 (∼88 % of total) transcripts and 1,301
(∼62 % of total) proteins, whose gradient migration patterns were successfully reconstructed, and thereby represents the first RNA/protein complexome resource
of a Gram-positive organism. The dataset readily verified many conserved major
complexes for the first time in S. pneumoniae and led to the discovery of a specific
interaction between the 3’!5’ exonuclease Cbf1 and the competence-regulating ciadependent sRNAs (csRNAs). Unexpectedly, trimming of the csRNAs by Cbf1 stabilized the former, thereby promoting their inhibitory function. cbf1 was further shown
to be part of the late competence genes and as such to act as a negative regulator of
competence.
Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development – gamete
formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins
are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the
mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized
to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp
proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript
levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein
complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had
been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively
expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the
P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability
of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages
becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs.
The biogenesis of spliceosomal UsnRNPs is a highly elaborate cellular process that occurs both in the nucleus and the cytoplasm. A major part of the process is the assembly of the Sm-core particle, which consists of a ring shaped heptameric unit of seven Sm proteins (SmD1•D2•F•E•G•D3•B) wrapped around a single stranded RNA motif (termed Sm-site) of spliceosomal UsnRNAs. This process occurs mainly in the cytoplasm by the sequential action of two biogenesis factors united in PRMT5- and SMN-complexes, respectively. The PRMT5-complex composed of the three proteins PRMT5, WD45 and pICln is responsible for the symmetric dimethylation of designated arginine residues in the C-terminal tails of some Sm proteins. The action of the PRMT5- complex results in the formation of assembly incompetent Sm-protein intermediates sequestered by the assembly chaperone pICln (SmD1•D2•F•E•G•pICln and pICln•D3•B). Due to the action of pICln, the Sm proteins in these complexes fail to interact with UsnRNAs to form the mature Sm-core. This kinetic trap is relieved by the action of the SMN-complex, which removes the pICln subunit and facilitates the binding of the Sm-core intermediates to the UsnRNA, thus forming the mature Sm-core particle. The human SMN complex consists of 9 subunits termed SMN, Gemin2-8 and Unrip. So far, there are no available atomic structures of the whole SMN-complex, but structures of isolated domains and subunits of the complex have been reported by several laboratories in the past years. The lack of structural information about the entire SMN complex most likely lies in the biophysical properties of the SMN complex, which possesses an oligomeric SMN core, and many unstructured and flexible regions. These were the biggest roadblocks for its structural elucidation using traditional methods such as X-ray crystallography, NMR or CryoEM. To circumvent these obstacles and to obtain structural insight into the SMN-complex, the Schizosaccharomyces pombe SMN complex was used as a model system in this work. In a collaboration with the laboratory of Dr. Remy Bordonne (IGMM, CNRS, France), we could show that the SpSMN complex is minimalistic in its composition, consisting only of SpSMN, SpGemin2, SpGemin8, SpGemin7 and SpGemin6. Using biochemical experiments, an interaction map of the SpSMN complex was established which was found to be highly similar to the reported map of the human SMN complex. The results of this study clearly show that SpSMN is the oligomeric core of the complex and provides the binding sites for the rest of the subunits. Through biochemical and X-ray scattering experiments, the properties of the SpSMN subunit such as oligomerization viii and intrinsic disorder, were shown to determine the overall biophysical characteristics of the whole complex. The structural basis of SpSMN oligomerization is presented in atomic detail which establishes a dimeric SpSMN as the fundamental unit of higher order SpSMN oligomers. In addition to oligomerization, the YG-box domain of SpSMN serves as the binding site for SpGemin8. The unstructured region of SpSMN imparts an unusual large hydrodynamic size, intrinsic disorder, and flexibility to the whole complex. Interestingly, these biophysical properties are partially mitigated by the presence of SpGemin8•SpGemin7•SpGemin6 subunits. These results classify the SpSMN complex as a multidomain entity connected with flexible linkers and characterize the SpSMN subunit to be the central oligomeric structural organizer of the whole complex.