Cell News | Issue 01, 2017 - page 31

Cell News 01/2017
Analysis of keratin mutations that render
the cytoskeleton thermosensitive
Melanie Homberg, Frederik Tellkamp, Carien Niessen and Thomas M. Magin
Presenting author: Melanie Homberg
University of Leipzig, Institute of Biology & SIKT,
Philipp-Rosenthal-Straße 55, 04103 Leipzig
Keratins (K) constitute the major cytoskeletal system of
epidermal keratinocytes and protect the epidermis against
mechanical force by forming extensive protein interaction net-
works. The occurrence of missense mutations in keratin genes
encoding K5 or K14 which cause the skin fragility disorder
epidermolysis bullosa simplex (EBS) has helped to identify ami-
no acid residues crucial for filament formation and stability.
Some mutations cause severe EBS accompanied by extensive
collapse of the keratin cytoskeleton into cytoplasmic protein
aggregates, while other mutations cause milder forms of the
disease with increased skin blistering during warmer summer
months. This indicates different disease mechanisms and the
involvement of environmental factors; however it is so far not
understood how elevated temperature cooperates with keratin
mutations to cause skin blistering.
Here, we present data that indicate the occurrence of hy-
per-phosphorylation in combination with K5 mutations that be-
have in a temperature sensitive fashion. Next to severe aggre-
gate forming mutations, we identified K5 and K14 mutations
that permit formation of normal appearing keratin filaments
under standard cell culture conditions. Interestingly, some of
these mutations display extensive aggregate formation upon
exposure to elevated temperature. The reversible and rapid
nature of keratin aggregation upon heat stress suggested the
involvement of posttranslational modifications. Phospho-pro-
teomic analysis of total cell proteins indicates that both - the
presence of a specific K5 mutation and keratin aggregation
upon exposure to higher temperature - are accompanied by el-
evated Ser phosphorylation of K5. To identify kinases involved,
kinase prediction tools and specific inhibitors are currently
investigated. In the absence of a full atomic structure, our data
raise the question how certain mutations mediate thermosen-
sitivity and phosphorylation of distinct sites.
Keratin-dependent regulation of mitochondrial activity
Kristin Jahn and Thomas M. Magin
Presenting author: Kristin Jahn
Institute of Biology, Division of Cell and Developmental Biology
and SIKT, University of Leipzig, 04103 Leipzig, Germany
The epidermis is a stratified epithelium which undergoes a
continuous cycle of proliferation and differentiation from
stem cells that give rise to differentiated keratinocytes. During
this cycle and during wounding/regeneration, keratinocytes
experience various metabolic and structural requirements to
which they must adapt. Differential expression of cytoskeletal
keratins is ideally suited to adapt keratinocytes to mechanical
and other forms of stress. Recently, we and others have found
that beyond mechanical protection, keratins participate in the
regulation of cell growth, including protein biosynthesis and
mitochondrial activity. In contrast to other tissues, the role of
mitochondria for epidermal differentiation, wound healing and
in epidermal disease, is not well understood.
We have recently found an altered distribution of mitochon-
dria in the skin of mice lacking all keratins. Corresponding
keratinocytes showed an increased expression of subunits of
the electron transport chain (ETC) complexes I and IV. Addi-
tionally, an altered composition of mitochondrial lipids with
highly increased cardiolipin content and an increased oxygen
consumption rate and therefore an increased ATP content was
demonstrated. Given that tissue regeneration is impaired in
several skin disorders caused by missense mutations in epider-
mal keratin genes, this suggests that keratin isotypes may be
involved in cell-specific and context-dependent regulation of
mitochondrial activity. To examine this, mitochondrial protein
and lipid composition and activity will be analyzed in kerati-
nocytes derived from epidermolysis bullosa simplex patients,
carrying K14 mutations. During the poster presentation, data
on electron transport chain complex (ETC) composition, the
formation of ETC supercomplexes, the oxygen consumption and
also the cellular localization of mitochondria will be discussed.
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