The complete amino acid sequence of the 61-kDa calmodulin-dependent, cyclic nucleotide phosphodiesterase (CaM-PDE) from bovine brain has been determined. The native protein is a homodimer of N alpha-acetylated, 529-residue polypeptide chains, each of which has a calculated molecular weight of 60,755. The structural organization of this CaM-PDE has been investigated with use of limited proteolysis and synthetic peptide analogues. A site capable of interacting with CaM has been identified, and the position of the catalytic domain has been mapped. A fully active, CaM-independent fragment (Mr = 36,000), produced by limited tryptic cleavage in the absence of CaM, represents a functional catalytic domain. N-Terminal sequence and size indicate that this 36-kDa fragment is comprised of residues 136 to approximately 450 of the CaM-PDE. This catalytic domain encompasses a approximately 250 residue sequence that is conserved among PDE isozymes of diverse size, phylogeny, and function. CaM-PDE and its PDE homologues comprise a unique family of proteins, each having a catalytic domain that evolved from a common progenitor. A search of the sequence for potential CaM-binding sites revealed only one 15-residue segment with both a net positive charge and the ability to form an amphiphilic alpha-helix. Peptide analogues that include this amphiphilic segment were synthesized. Each was found to inhibit the CaM-dependent activation of the enzyme and to bind directly to CaM with high affinity in a calcium-dependent manner. This site is among the sequences cleaved from a 45-kDa chymotryptic fragment that has the complete catalytic domain but no longer binds CaM. These results indicate that residues located between position 23 and 41 of the native enzyme contribute significantly to the binding of CaM although the involvement of residues from additional sites is not excluded.
Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (T
H
) 1 and T
H
17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.
Case-based, interactive sessions for small groups (in a large medical school class of 150 students) reinforces basic immunology concepts by including clinical scenarios that stimulate student learning and consolidate critical concepts. Careful design of cases (designing backwards from the key concepts) leads students through successively more complicated and linked group-work questions. This paper details why cases are effective learning tools, how to design an effective case, how to ask appropriate questions and how to help students apply basic immunology concepts to a case. Each group work session is facilitated and followed by a question and answer presentation by faculty, where student groups are directly asked to answer the questions and also challenged with "bonus questions" not presented with the original case. This allows students to "put together" immunology information into a "story" that they can tell and prevents student frustration by summarizing the results at the end of each case. Case design is carefully discussed including clinical relevancy and accuracy, how to write questions that do not give away the answers, how to emphasize mechanistic questions that allow students to "clinically explain as a physician" the immunological basis for the answers. Additionally, students better understand the role of immunity in both normal and disease states. A case-based approach promotes student learning by re-emphasizing basic concepts in the context of the case and promotes better students understanding of critical immunological concepts.
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