Numaswitch: an efficient high-titer expression platform to produce peptides and small proteins.

The production of peptides as active pharmaceutical ingredients (APIs) by recombinant technologies is of emerging interest. A reliable production platform, however, is still missing due the inherent characteristics of peptides such as proteolytic sensitivity, aggregation and cytotoxicity. We have developed a new technology named Numaswitch solving present limitations. Numaswitch was successfully employed for the production of diverse peptides and small proteins varying in length, physicochemical and functional characteristics, including Teriparatide, Linaclotide, human ß-amyloid and Serum amyloid A3. Additionally, the potential of Numaswitch for a cost-efficient commercial production is demonstrated yielding > 2 g Teriparatide per liter fermentation broth in a quality meeting API standard.

NCAM140 is translocated into the nucleus by an importin-ß1-dependent mechanism.

The neural cell adhesion molecule (NCAM) is important for neural development and for plasticity in adult brain. Previous studies demonstrated a calmodulin-dependent import of a transmembrane fragment of NCAM into the nucleus that regulates gene expression. In a protein macroarray we identified importin-ß1 as a potential interaction partner of NCAM's cytoplasmic tail. The interaction was verified and an importin-ß1-dependent import of NCAM into the nucleus could be demonstrated using quantitative immunofluorescence analysis. Generation of NCAM deletion mutants revealed that the last amino acids of the cytoplasmic region of NCAM are dispensable whereas other parts of NCAM's cytoplasmic tail take part in its nuclear translocation. With this study we propose an alternative nuclear route for NCAM via the classical importin-mediated import.

Cell Adhesion Molecules and Ubiquitination-Functions and Significance.

Cell adhesion molecules of the immunoglobulin (Ig) superfamily represent the biggest group of cell adhesion molecules. They have been analyzed since approximately 40 years ago and most of them have been shown to play a role in tumor progression and in the nervous system. All members of the Ig superfamily are intensively posttranslationally modified. However, many aspects of their cellular functions are not yet known. Since a few years ago it is known that some of the Ig superfamily members are modified by ubiquitin. Ubiquitination has classically been described as a proteasomal degradation signal but during the last years it became obvious that it can regulate many other processes including internalization of cell surface molecules and lysosomal sorting. The purpose of this review is to summarize the current knowledge about the ubiquitination of cell adhesion molecules of the Ig superfamily and to discuss its potential physiological roles in tumorigenesis and in the nervous system.

Kinesin-1 promotes post-Golgi trafficking of NCAM140 and NCAM180 to the cell surface.

The neural cell adhesion molecule (NCAM, also known as NCAM1) is important during neural development, because it contributes to neurite outgrowth in response to its ligands at the cell surface. In the adult brain, NCAM is involved in regulating synaptic plasticity. The molecular mechanisms underlying delivery of NCAM to the neuronal cell surface remain poorly understood. We used a protein macroarray and identified the kinesin light chain 1 (KLC1), a component of the kinesin-1 motor protein, as a binding partner of the intracellular domains of the two transmembrane isoforms of NCAM, NCAM140 and NCAM180. KLC1 binds to amino acids CGKAGPGA within the intracellular domain of NCAM and colocalizes with kinesin-1 in the Golgi compartment. Delivery of NCAM180 to the cell surface is increased in CHO cells and neurons co-transfected with kinesin-1. We further demonstrate that the p21-activated kinase 1 (PAK1) competes with KLC1 for binding to the intracellular domain of NCAM and contributes to the regulation of the membrane insertion of NCAM. Our results indicate that NCAM is delivered to the cell surface through a kinesin-1-mediated transport mechanism in a PAK1-dependent manner.

Cytoplasmic domain of NCAM140 interacts with ubiquitin-fold modifier-conjugating enzyme-1 (Ufc1).

The neural cell adhesion molecule NCAM is implicated in different neurodevelopmental processes and in synaptic plasticity in adult brain. The cytoplasmic domain of NCAM interacts with several cytoskeletal proteins and signaling molecules. To identify novel interaction partners of the cytosolic domain of NCAM a protein macroarray has been performed. We identified the ubiquitin-fold modifier-conjugating enzyme-1 (Ufc1) as an interaction partner of NCAM140. Ufc1 is one of the enzymes involved in modification of proteins with the ubiquitin-like molecule ubiquitin-fold modifier-1 (Ufm1). We also observed a partial co-localization of NCAM140 with Ufc1 and Ufm1 and increased endocytosis of NCAM140 in the presence of Ufm1 suggesting a possible ufmylation of NCAM140 and a potential novel function of Ufm1 for cell surface proteins.

UCHL1 regulates ubiquitination and recycling of the neural cell adhesion molecule NCAM.

The neural cell adhesion molecule (NCAM) is involved in neural development and in plasticity in the adult brain. NCAM140 and NCAM180 isoforms are transmembrane proteins with cytoplasmic domains that differ only in an alternatively spliced exon in the NCAM180 isoform. Both isoforms can interact with several extracellular and cytoplasmic molecules mediating NCAM-dependent functions. Most identified intracellular interaction partners bind to both isoforms, NCAM140 and NCAM180. To identify further intracellular interaction partners specifically binding to NCAM180 the cytosolic domain of human NCAM180 was recombinantly expressed and applied onto a protein macroarray containing the protein library from human fetal brain. We identified the ubiquitin C-terminal hydrolase (UCHL1) which has been described as a de-ubiquitinating enzyme as a potential interaction partner of NCAM180. Since NCAM180 and NCAM140 are ubiquitinated, NCAM140 was included in the subsequent experiments. A partial colocalization of both NCAM isoforms and UCHL1 was observed in primary neurons and the B35 neuroblastoma cell line. Overexpression of UCHL1 significantly decreased constitutive ubiquitination of NCAM180 and NCAM140 whereas inhibition of endogenous UCHL1 increased NCAM's ubiquitination. Furthermore, lysosomal localization of NCAM180 and NCAM140 was significantly reduced after overexpression of UCHL1 consistent with a partial colocalization of internalized NCAM with UCHL1. These data indicate that UCHL1 is a novel interaction partner of both NCAM isoforms that regulates their ubiquitination and intracellular trafficking.