Mitochondrial protein quality control is crucial for the maintenance of correct mitochondrial homeostasis. It is ensured by several specific mitochondrial proteases located across the various mitochondrial subcompartments. Here, we focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 (AFG3 Like Matrix AAA Peptidase Subunit 2) and YME1L (YME1 like ATPase) of mitochondrial inner membrane AAA (ATPases Associated with diverse cellular Activities) complexes in the maintenance of mitochondrial structure and respiratory chain integrity. We demonstrate that loss of AFG3L2 and YME1L, both alone and in combination, results in diminished cell proliferation, fragmentation of mitochondrial reticulum, altered cristae morphogenesis, and defective respiratory chain biogenesis. The double AFG3L2/YME1L knockdown cells showed marked upregulation of OPA1 protein forms, with the most prominent increase in short OPA1 (optic atrophy 1). Loss of either protease led to marked elevation in OMA1 (OMA1 zinc metallopeptidase) (60 kDa) and severe reduction in the SPG7 (paraplegin) subunit of the m-AAA complex. Loss of the YME1L subunit led to an increased Drp1 level in mitochondrial fractions. While loss of YME1L impaired biogenesis and function of complex I, knockdown of AFG3L2 mainly affected the assembly and function of complex IV. Our results suggest cooperative and partly redundant functions of AFG3L2 and YME1L in the maintenance of mitochondrial structure and respiratory chain biogenesis and stress the importance of correct proteostasis for mitochondrial integrity.

• MeSH
• ATPázy spojené s různými buněčnými aktivitami genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• metaloendopeptidasy genetika   metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   ultrastruktura   MeSH
• proliferace buněk genetika   fyziologie   MeSH
• proteasy závislé na ATP genetika   metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy metabolismus   MeSH
• ATPázy spojené s různými buněčnými aktivitami metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA metabolismus   MeSH
• elektronová kryomikroskopie MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• replikace DNA * MeSH
• replikační protein C chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Pathogenic variants in the ATAD3A gene lead to a heterogenous clinical picture and severity ranging from recessive neonatal-lethal pontocerebellar hypoplasia through milder dominant Harel-Yoon syndrome up to, again, neonatal-lethal but dominant cardiomyopathy. The genetic diagnostics of ATAD3A-related disorders is also challenging due to three paralogous genes in the ATAD3 locus, making it a difficult target for both sequencing and CNV analyses. RESULTS: Here we report four individuals from two families with compound heterozygous p.Leu77Val and exon 3-4 deletion in the ATAD3A gene. One of these patients was characterized as having combined OXPHOS deficiency based on decreased complex IV activities, decreased complex IV, I, and V holoenzyme content, as well as decreased levels of COX2 and ATP5A subunits and decreased rate of mitochondrial proteosynthesis. All four reported patients shared a strikingly similar clinical picture to a previously reported patient with the p.Leu77Val variant in combination with a null allele. They presented with a less severe course of the disease and a longer lifespan than in the case of biallelic loss-of-function variants. This consistency of the phenotype in otherwise clinically heterogenous disorder led us to the hypothesis that the severity of the phenotype could depend on the severity of variant impact. To follow this rationale, we reviewed the published cases and sorted the recessive variants according to their impact predicted by their type and the severity of the disease in the patients. CONCLUSION: The clinical picture and severity of ATAD3A-related disorders are homogenous in patients sharing the same combinations of variants. This knowledge enables deduction of variant impact severity based on known cases and allows more accurate prognosis estimation, as well as a better understanding of the ATAD3A function.

• MeSH
• ATPázy spojené s různými buněčnými aktivitami * genetika   MeSH
• biologická variabilita populace * MeSH
• fenotyp MeSH
• lidé MeSH
• mitochondrie * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.

• MeSH
• ATPázy spojené s různými buněčnými aktivitami genetika   metabolismus   MeSH
• buněčné linie MeSH
• chromatin enzymologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• ftalaziny farmakologie   MeSH
• kur domácí MeSH
• mutageny toxicita   MeSH
• nádorové buněčné linie MeSH
• nestabilita genomu MeSH
• PARP inhibitory farmakologie   MeSH
• piperaziny farmakologie   MeSH
• poly(ADP-ribosa)polymerasa 1 metabolismus   MeSH
• poškození DNA * MeSH
• protinádorové látky farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM.

• Publikační typ
• časopisecké články MeSH

• MeSH
• arginin analýza   MeSH
• běloši genetika   MeSH
• DNA genetika   MeSH
• faktor VIII genetika   MeSH
• glutamáty analýza   MeSH
• hemofilie A epidemiologie   genetika   MeSH
• kyselina glutamová MeSH
• lidé MeSH
• lysin analýza   MeSH
• polymerázová řetězová reakce MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• Geografické názvy
• Československo MeSH

• MeSH
• arginin * analýza   MeSH
• běloši * genetika   MeSH
• DNA genetika   MeSH
• faktor VIII * genetika   MeSH
• glutamáty * analýza   MeSH
• hemofilie A epidemiologie   genetika   MeSH
• kyselina glutamová MeSH
• lidé MeSH
• lysin * analýza   MeSH
• polymerázová řetězová reakce MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• Geografické názvy
• Československo MeSH

BACKGROUND: Endovascular aneurysm repair (EVAR) has created new possibilities for patients with abdominal aortic aneurysms (AAAs), and in recent years it has become tremendously popular. Use of EVAR in selected groups of patients allows mortality and morbidity to be reduced in comparison to open repair. However, complications such as endoleaks (ELs) can be of great concern and warrant urgent therapy to prevent sac rupture. CASE PRESENTATION: The case report presents urgent endovascular treatment of a high-risk type IA EL in a polymorbid 68-year-old patient 7 years after primary EVAR. The principle of treatment was parallel implantation of the proximal SG extension with the renal SG into the right renal artery (chimney technique). The subsequent type II collateral EL was treated by direct transabdominal AAA sac puncture and thrombin embolization. CONCLUSION: EL can be a cause for urgent intervention, but specific anatomic features often require specialized SG types which are not readily available. The chimney technique allows the use of immediately available stent grafts to address endoleak in the setting of impending abdominal aneurysm rupture.

• Publikační typ
• časopisecké články MeSH

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

• MeSH
• apoptóza MeSH
• genový knockdown MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• lidé MeSH
• metaloendopeptidasy metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• NADH, NADPH oxidoreduktasy metabolismus   MeSH
• proliferace buněk MeSH
• proteasy závislé na ATP metabolismus   MeSH
• proteasy metabolismus   MeSH
• protein - isoformy metabolismus   MeSH
• respirační komplex IV metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   MeSH
• transport elektronů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Svůj příspěvek jsem zpracovala na téma: „Canisterapie z pohledu terapeuta“. V Domově pro seniory Máj, kde pracuji jako ergoterapeut, je realizována canisterapie návštěvního typu. Při práci s klienty jsou v našem zařízení využívány metody AAA a AAT. Canisterapie je indikována převážně klientům s Alzheimerovou demencí a klientům po cévních mozkových příhodách. Ergoterapie je součástí komplexní rehabilitace. Canisterapie práci ergoterapeuta nejen usnadňuje, ale především doplňuje, neboť obě terapie mají společné cíle. Ergoterapie, stejně jako canisterapie, umožňuje vzájemnou interakci, přispívá k aktivizaci, lze ji uplatnit při zlepšování dovedností jemné motoriky a při ovlivňování psychické kondice klientů. Při porovnání metod AAA a AAT spatřuji metodu individuální canisterapie jako účelnější, neboť umožňuje vycházet z individuálních potřeb klienta a poskytuje více prostoru pro ovlivňování postižených či oslabených funkcí volbou vhodných canisterapeutických aktivit. Canisterapie je v našem zařízení obyvateli přijímána kladně a zvyšuje jejich chuť účastnit se i dalších aktivit. Hlavním důvodem nabídky volnočasových aktivit a terapií obyvatelům našeho domova je dosažení co nejvyšší kvality života, zvýšení sebevědomí a prožití důstojného stáří.

I compiled my contribution named “Canistherapy from the viewpoint of therapist” in the Old people´s home May, where I work as an ergotherapist and where outpatient type canistherapy is practised. AAA and AAT methods are employed in the work with clients in our facility. Canistherapy is prevalently indicated for clients with Alzhemimer dementia and clients after cerebrovascular events. Ergotherapy is a part of complex rehabilitation. Canistherapy not only facilitates but particularly supplements the ergotherapist work, since both types of the therapy are directed to common targets. Ergotherapy, similarly as canistherapy, makes possible mutual interactions, contributes to the activization and may be implemented for improving fine motor skills and for affecting the mental condition of clients. In a comparison of AAA and AAT methods, I consider the individual canistherapy method as more purposeful, since it makes it possible to start from individual needs of the clients and provides more space for affecting involved or weakened functions through the choice of suitable canistherapeutic activities. In our facility, canistherapy is positively accepted by the clients and it enhances their interest in participation in further activities, too. The main reason for the offer of free time activities and therapies to clients from our home is to achieve as high quality of life as possible, enhance the self-confidence and enjoy the worthy old age.

• MeSH
• domovy pro seniory využití   MeSH
• ergoterapie metody   využití   MeSH
• geriatrické ošetřovatelství metody   MeSH
• kvalitativně upravené roky života MeSH
• lidé MeSH
• psi psychologie   MeSH
• senioři fyziologie   psychologie   MeSH
• volnočasové aktivity psychologie   MeSH
• výsledek terapie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi psychologie   MeSH
• senioři fyziologie   psychologie   MeSH
• zvířata MeSH